Families of non-cross-hybridizing polynucleotides for use as tags and tag complements, manufacture and use thereof

ABSTRACT

A family of minimally cross-hybridizing nucleotide sequences, methods of use, etc. A specific family of 1168 24mers is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/470,073, which is the U.S. national stage of international patentapplication PCT/CA02/00089, filed Jan. 25, 2002, which claims thebenefit of and priority under 35 U.S.C. §119(e) to U.S. ProvisionalPatent Application Nos. 60/263,710, filed Jan. 25, 2001, and 60/303,799,filed Jul. 10, 2001, the entire disclosures of each of which are hereinincorporated by reference.

FIELD OF THE INVENTION

This invention relates to families of oligonucleotide tags for use, forexample, in sorting molecules. Members of a given family of tags can bedistinguished one from the other by specific hybridization to their tagcomplements.

BACKGROUND OF THE INVENTION

Specific hybridization of oligonucleotides and their analogs is afundamental process that is employed in a wide variety of research,medical, and industrial applications, including the identification ofdisease-related polynucleotides in diagnostic assays, screening forclones of novel target polynucleotides, identification of specificpolynucleotides in blots of mixtures of polynucleotides, therapeuticblocking of inappropriately expressed genes and DNA sequencing. Sequencespecific hybridization is critical in the development of high throughputmultiplexed nucleic acid assays. As formats for these assays expand toencompass larger amounts of sequence information acquired throughprojects such as the Human Genome project, the challenge of sequencespecific hybridization with high fidelity is becoming increasinglydifficult to achieve.

In large part, the success of hybridization using oligonucleotidesdepends on minimizing the number of false positives and false negatives.Such problems have made the simultaneous use of multiple hybridizationprobes in a single experiment i.e. multiplexing, particularly in theanalysis of multiple gene sequences on a gene microarray, verydifficult. For example, in certain binding assays, a number of nucleicacid molecules are bound to a chip with the desire that a given “target”sequence will bind selectively to its complement attached to the chip.Approaches have been developed that involve the use of oligonucleotidetags attached to a solid support that can be used to specificallyhybridize to the tag complements that are coupled to probe sequences.Chetverin et al. (WO 93/17126) uses sectioned, binary oligonucleotidearrays to sort and survey nucleic acids. These arrays have a constantnucleotide sequence attached to an adjacent variable nucleotidesequence, both bound to a solid support by a covalent linking moiety.These binary arrays have advantages compared with ordinary arrays inthat they can be used to sort strands according to their terminalsequences so that each strand binds to a fixed location on an array. Thedesign of the terminal sequences in this approach comprises the use ofconstant and variable sequences. U.S. Pat. Nos. 6,103,463 and 6,322,971issued to Chetverin et al. on Aug. 15, 2000 and Nov. 27, 2001,respectively.

This concept of using molecular tags to sort a mixture of molecules isanalogous to molecular tags developed for bacterial and yeast genetics(Hensel et al., Science; 269, 400-403: 1995 and Schoemaker et al.,Nature Genetics; 14, 450-456: 1996). Here, a method termed “signaturetagged” mutagenesis in which each mutant is tagged with a different DNAsequence is used to recover mutant genes from a complex mixture ofapproximately 10,000 bacterial colonies. In the tagging approach ofBarany et al. (WO 9731256), known as the “zip chip”, a family of nucleicacid molecules, the “zip-code addresses”, each different from eachother, are set out on a grid. Target molecules are attached tooligonucleotide sequences complementary to the “zipcode addresses,”referred to as “zipcodes,” which are used to specifically hybridize tothe address locations on the grid. While the selection of these familiesof polynucleotide sequences used as addresses is critical for correctperformance of the assay, the performance has not been described.

Working in a highly parallel hybridization environment requiringspecific hybridization imposes very rigorous selection criteria for thedesign of families of oligonucleotides that are to be used. The successof these approaches is dependent on the specific hybridization of aprobe and its complement. Problems arise as the family of nucleic acidmolecules cross-hybridize or hybridize incorrectly to the targetsequences. While it is common to obtain incorrect hybridizationresulting in false positives or an inability to form hybrids resultingin false negatives, the frequency of such results must be minimized. Inorder to achieve this goal certain thermodynamic properties of formingnucleic acid hybrids must be considered. The temperature at whicholigonucleotides form duplexes with their complementary sequences knownas the T_(m) (the temperature at which 50% of the nucleic acid duplex isdissociated) varies according to a number of sequence dependentproperties including the hydrogen bonding energies of the canonicalpairs A-T and G-C (reflected in GC or base composition), stacking freeenergy and, to a lesser extent, nearest neighbour interactions. Theseenergies vary widely among oligonucleotides that are typically used inhybridization assays. For example, hybridization of two probe sequencescomposed of 24 nucleotides, one with a 40% GC content and the other witha 60% GC content, with its complementary target under standardconditions theoretically may have a 10° C. difference in meltingtemperature (Mueller et al., Current Protocols in Mol. Biol.; 15, 5:1993). Problems in hybridization occur when the hybrids are allowed toform under hybridization conditions that include a single hybridizationtemperature that is not optimal for correct hybridization of alloligonucleotide sequences of a set. Mismatch hybridization ofnon-complementary probes can occur forming duplexes with measurablemismatch stability (Peyret et al., Biochemistry; 38: 3468-77, 1999).Mismatching of duplexes in a particular set of oligonucleotides canoccur under hybridization conditions where the mismatch results in adecrease in duplex stability that results in a higher T_(m) than theleast stable correct duplex of that particular set. For example, ifhybridization is carried out under conditions that favor the AT-richperfect match duplex sequence, the possibility exists for hybridizing aGC-rich duplex sequence that contains a mismatched base having a meltingtemperature that is still above the correctly formed AT-rich duplex.Therefore design of families of oligonucleotide sequences that can beused in multiplexed hybridization reactions must include considerationfor the thermodynamic properties of oligonucleotides and duplexformation that will reduce or eliminate cross hybridization behaviorwithin the designed oligonucleotide set.

The development of such families of tags has been attempted over theyears with varying degrees of success. There are a number of differentapproaches for selecting sequences for use in multiplexed hybridizationassays. The selection of sequences that can be used as zipcodes or tagsin an addressable array has been described in the patent literature inan approach taken by Brenner and co-workers. U.S. Pat. No. 5,654,413describes a population of oligonucleotide tags (and corresponding tagcomplements) in which each oligonucleotide tag includes a plurality ofsubunits, each subunit consisting of an oligonucleotide having a lengthof from three to six nucleotides and each subunit being selected from aminimally cross hybridizing set, wherein a subunit of the set would haveat least two mismatches with any other sequence of the set. Table II ofthe Brenner patent specification describes exemplary groups of 4mersubunits that are minimally cross hybridizing according to theaforementioned criteria. In the approach taken by Brenner, constructingnon cross-hybridizing oligonucleotides, relies on the use of subunitsthat form a duplex having at least two mismatches with the complement ofany other subunit of the same set. The ordering of subunits in theconstruction of oligonucleotide tags is not specifically defined.

Parameters used in the design of tags based on subunits are discussed inBarany et al. (WO 9731256). For example, in the design of polynucleotidesequences that are for example 24 nucleotides in length (24mer) derivedfrom a set of four possible tetramers in which each 24mer “address”differs from its nearest 24mer neighbour by 3 tetramers. They discussfurther that, if each tetramer differs from each other by at least twonucleotides, then each 24mer will differ from the next by at least sixnucleotides. This is determined without consideration for insertions ordeletions when forming the alignment between any two sequences of theset. In this way a unique “zip code” sequence is generated. The zip codeis ligated to a label in a target dependent manner, resulting in aunique “zip code” which is then allowed to hybridize to its address onthe chip. To minimize cross-hybridization of a “zip code” to other“addresses”, the hybridization reaction is carried out at temperaturesof 75-80° C. Due to the high temperature conditions for hybridization,24mers that have partial homology hybridize to a lesser extent thansequences with perfect complementarity and represent ‘dead zones’. Thisapproach of implementing stringent hybridization conditions for example,involving high temperature hybridization, is also practiced by Brenneret. al.

The current state of technology for designing non-cross hybridizing tagsbased on subunits does not provide sufficient guidance to construct afamily of relatively large numbers of sequences with practical value inassays that require stringent non-cross hybridizing behavior.

A multiplex sequencing method has been described in U.S. Pat. No.4,942,124, which issued to Church on Jul. 17, 1990. The method requiresat least two vectors which differ from each other at a tag sequence. Itis stated that a tag sequence in one vector will not hybridize understringent hybridization conditions to a tag sequence (i.e.,complementary probes do not cross-hybridize) in another vector.Exemplary stringent hybridization conditions are given as 42° C. in500-1000 mM sodium phosphate buffer. A set of 42 20-mer tag sequences,all of which lack G residues, is given in FIG. 3 of the specification.Details of how the sequences were obtained are not provided, althoughChurch states that initially 92 were chosen on the basis of their havingsufficient sequence diversity to insure uniqueness.

So while it is possible for a person knowledgeable in the field todesign a small number of non-cross hybridizing tags, it is difficult todesign a larger number such tags. A co-pending application of the ownerof this patent application describes such a set of 210 non-crosshybridizing tags that have a practical value. A method described ininternational patent application No. PCT/CA 01/00141 published under WO01/59151 on Aug. 16, 2001. Little guidance is provided, however, for theprovision of a larger set, say 1000 or so, of non-cross hybridizingtags. Since having sets of approximately 1000 non-cross hybridizingtags, or more, would be of considerable practical value, it would beuseful to develop such a set.

Thus, while it is desirable with such arrays to have, at once, a largenumber of address molecules, the address molecules should each be highlyselective for its own complement sequence: While such an array providesthe advantage that the family of molecules making up the grid isentirely of design, and does not rely on sequences as they occur innature, the provision of a family of molecules, which is sufficientlylarge and where each individual member is sufficiently selective for itscomplement over all the other zipcode molecules (i.e., where there issufficiently low cross-hybridization, or cross-talk) continues to eluderesearchers.

SUMMARY OF INVENTION

A family of 1168 sequences was obtained using a computer algorithm tohave desirable hybridization properties for use in nucleic aciddetection assays. The sequence set of 1168 oligonucleotides waspartially characterized in hybridization assays, demonstrating theability of family members to correctly hybridize to their complementarysequences with minimal cross hybridization. These are the sequenceshaving SEQ ID NOs:1 to 1168 of Table I.

Variant families of sequences (seen as tags or tag complements) of afamily of sequences taken from Table I are also part of the invention.For the purposes of discussion, a family or set of oligonucleotides willoften be described as a family of tag complements, but it will beunderstood that such a set could just easily be a family of tags.

A family of complements is obtained from a set of oligonucleotides basedon a family of oligonucleotides such as those of Table I. To simplifydiscussion, providing a family of complements based on theoligonucleotides of Table I will be described.

Firstly, the groups of sequences based on the oligonucleotides of TableI can be represented as shown in Table IA.

TABLE IA Numeric sequences corresponding to nucleotide base patterns ofa set of oligonucleotides Sequence Numeric Pattern Identifier 1 1 1 2 23 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 12 2 2 3 1 2 3 1 2 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 3 2 31 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 4 2 2 2 3 2 3 2 1 3 1 1 2 12 3 2 3 2 2 3 2 2 1 1 5 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 16 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 7 3 2 3 2 2 2 1 2 3 22 1 2 1 2 3 2 3 1 1 3 2 2 2 8 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 11 3 2 9 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 10 1 2 3 1 3 1 11 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 11 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 32 1 3 2 2 2 12 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 13 1 1 13 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 14 3 2 1 3 1 1 1 2 1 3 2 2 2 12 2 3 1 2 3 1 2 2 3 15 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 216 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 17 1 2 1 1 3 2 2 1 21 1 3 2 3 2 2 1 2 3 2 3 1 3 2 18 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 23 2 2 3 19 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 20 1 1 3 2 13 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 21 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 11 3 1 2 2 3 1 2 22 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 23 31 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 24 2 1 3 1 2 3 1 3 1 2 2 11 3 2 3 2 2 1 2 2 2 3 1 25 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 13 26 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 27 3 2 3 1 1 2 3 12 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 28 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 31 2 2 1 3 29 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 30 2 1 2 12 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 31 2 2 1 1 3 2 3 2 2 1 3 2 2 1 22 2 3 2 2 3 2 1 3 32 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 332 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 34 2 2 3 2 1 3 1 2 2 1 31 2 3 2 3 2 2 2 3 2 1 1 1 35 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 22 2 36 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 37 2 3 2 3 1 3 11 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 38 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 13 1 3 2 2 3 39 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 40 2 1 31 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 41 3 2 2 1 2 3 1 2 3 2 3 2 1 21 1 3 2 1 1 2 1 2 3 42 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 343 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 44 3 2 3 1 3 1 2 1 21 3 1 2 2 2 1 3 1 1 1 3 2 1 1 45 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 21 3 2 1 46 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 47 1 2 2 2 32 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 48 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 13 1 2 1 1 3 1 1 49 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 50 32 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 51 2 2 2 3 2 3 1 1 3 1 2 31 1 3 2 1 2 2 2 3 2 1 2 52 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 21 53 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 54 3 2 2 1 2 2 2 32 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 55 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 31 1 2 1 3 56 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 57 1 2 2 32 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 58 2 3 1 3 1 1 2 3 2 1 1 1 3 1 12 3 2 2 2 1 2 2 3 59 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 603 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 61 3 1 2 2 3 1 2 1 2 2 23 1 1 2 3 2 2 2 3 2 2 2 3 62 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 21 2 63 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 64 1 1 1 2 2 2 31 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 65 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 22 3 2 2 3 2 66 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 67 1 2 32 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 68 2 3 2 2 2 1 1 1 3 1 2 3 1 22 3 1 1 3 1 1 1 2 3 69 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 170 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 71 1 2 1 3 1 2 3 2 11 3 1 3 1 1 1 2 2 3 2 3 1 1 1 72 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 31 3 2 1 73 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 74 1 1 2 3 21 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 75 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 12 3 2 2 3 1 3 2 76 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 77 31 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 78 2 1 2 2 3 2 1 3 1 1 3 21 1 1 3 2 2 1 3 1 1 3 2 79 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 23 80 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 81 1 2 1 1 3 1 1 12 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 82 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 31 2 1 2 1 83 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 84 3 1 2 21 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 85 2 1 2 3 2 3 2 3 2 2 3 2 2 2 13 2 3 2 2 1 2 2 1 86 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 873 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 88 2 1 3 2 1 2 2 1 3 2 11 3 2 1 2 3 2 3 1 2 2 3 2 89 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 11 2 90 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 91 3 1 2 2 3 1 12 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 92 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 21 1 3 2 2 1 93 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 94 2 2 21 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 95 3 1 2 1 3 1 2 2 2 1 3 1 1 23 1 1 2 2 1 1 3 2 3 96 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 197 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 98 2 3 2 2 2 1 2 3 21 3 2 3 2 1 3 1 2 2 3 1 1 2 2 99 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 21 3 2 1 100 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 101 2 1 3 22 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 102 3 2 1 1 2 3 1 2 1 1 2 3 1 13 2 3 2 1 2 1 2 1 3 103 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1104 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 105 2 1 1 2 3 1 1 31 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 106 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 21 3 2 2 1 3 107 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 108 3 11 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 109 1 3 2 2 1 2 1 1 3 2 2 23 2 3 1 3 1 1 2 2 1 1 3 110 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 23 2 111 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 112 3 1 3 2 2 11 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 113 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 13 2 1 2 1 2 1 3 114 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 1152 1 2 3 2 1 1 1 3 1 1 1 3 2 3 1 1 1 3 1 1 3 1 1 116 2 3 1 1 2 3 2 1 3 11 1 2 3 1 1 2 3 2 2 3 1 1 1 117 1 1 2 2 3 1 1 2 1 3 2 3 2 3 2 3 1 3 2 22 1 1 2 118 1 3 1 2 1 2 2 3 2 2 2 3 1 2 2 1 1 2 3 1 1 3 1 3 119 1 1 1 32 2 3 2 1 1 1 3 2 2 3 1 1 3 1 2 1 1 1 3 120 3 2 2 1 1 3 1 3 1 2 2 1 2 31 3 1 2 3 2 1 2 2 1 121 1 3 1 1 3 1 2 1 2 1 1 3 1 1 3 1 2 2 3 1 1 2 2 3122 3 2 1 3 1 1 1 2 2 2 3 1 1 2 2 3 1 2 3 2 3 1 1 1 123 1 1 3 1 3 2 1 31 2 2 3 1 2 1 1 3 2 1 2 1 2 3 1 124 2 3 1 2 1 2 1 3 2 1 3 2 3 1 1 3 1 11 2 1 1 3 2 125 1 3 1 2 1 1 2 3 1 2 3 1 3 1 1 1 2 3 1 1 3 1 2 1 126 1 23 2 3 1 1 1 3 2 1 2 2 2 3 2 3 1 2 1 2 1 3 2 127 1 1 2 1 1 3 1 3 1 1 2 23 1 2 1 2 3 1 1 3 1 2 3 128 2 1 1 3 2 3 2 1 2 2 2 1 3 2 1 3 1 1 2 3 1 13 2 129 2 1 2 3 2 2 1 3 1 2 2 2 3 2 2 3 1 3 1 2 2 3 1 2 130 1 3 2 2 2 32 1 2 3 1 1 3 1 3 1 2 1 3 2 1 2 2 2 131 3 1 3 1 1 1 2 3 2 2 1 2 3 2 1 22 2 1 3 2 1 3 2 132 2 1 2 3 2 3 1 3 1 1 2 3 2 3 2 2 2 3 1 2 2 2 1 1 1333 2 1 2 3 2 2 2 3 2 2 2 1 2 1 3 1 1 2 3 2 1 2 3 134 3 1 3 2 1 2 1 2 1 31 1 3 1 1 1 3 1 1 1 2 2 2 3 135 1 2 3 1 3 2 3 1 1 3 2 1 1 1 2 3 2 1 3 22 1 2 2 136 2 2 1 1 3 1 1 3 2 3 1 3 2 2 1 2 2 3 2 3 1 2 1 2 137 1 2 3 11 1 2 3 1 3 1 1 2 1 2 2 3 2 2 3 2 2 2 3 138 3 1 2 2 1 1 2 3 1 2 2 1 2 32 3 1 1 2 2 3 1 2 3 139 3 1 1 1 2 3 2 2 1 1 1 3 1 2 1 2 3 1 1 1 3 2 1 3140 2 1 2 2 3 2 2 3 1 2 2 2 3 1 2 1 2 2 1 3 2 3 2 3 141 2 2 2 1 2 3 2 22 3 2 3 2 1 2 3 2 1 1 3 2 1 3 2 142 1 1 2 2 3 1 1 1 3 1 1 2 2 3 2 3 2 31 1 2 2 3 1 143 2 3 1 3 2 2 2 3 1 1 2 2 2 3 2 2 2 3 1 3 2 1 1 2 144 3 12 3 2 1 2 1 1 2 3 1 2 3 2 3 2 3 2 1 1 1 2 2 145 1 2 3 2 3 1 3 1 3 1 1 31 1 2 2 2 3 2 2 2 1 2 2 146 3 2 3 1 2 1 1 1 3 2 1 2 2 3 2 2 3 1 2 1 3 11 1 147 3 1 1 3 2 1 3 1 1 2 1 3 1 1 1 3 2 2 1 1 2 1 3 1 148 2 2 3 2 3 21 3 2 2 1 1 3 1 3 2 2 3 2 2 2 1 1 2 149 2 1 3 2 1 3 2 1 1 3 2 2 3 2 2 13 1 1 2 1 3 2 2 150 1 1 2 2 2 3 1 1 3 2 1 2 1 1 2 3 1 1 2 3 2 3 2 3 1512 1 3 1 1 1 2 2 3 2 1 3 2 1 2 2 2 3 1 3 1 3 1 1 152 2 3 2 1 2 1 2 3 2 21 1 2 3 1 3 1 2 3 2 2 3 2 1 153 2 1 2 2 2 3 1 2 1 1 3 1 3 1 1 2 3 1 1 31 1 3 2 154 2 2 3 1 1 2 1 3 2 3 2 1 1 2 3 1 1 2 1 2 3 1 2 3 155 3 2 1 32 2 2 3 2 3 1 1 2 1 3 1 1 2 2 1 3 2 2 2 156 1 1 1 3 1 2 3 1 2 2 3 2 1 12 2 2 3 2 3 2 3 1 1 157 3 1 1 3 1 2 2 3 2 2 3 1 3 2 2 1 1 2 1 3 1 2 1 1158 1 3 1 2 2 1 2 3 2 1 3 2 3 1 2 3 2 1 1 1 2 3 2 2 159 3 1 1 2 2 2 1 31 2 3 2 1 3 1 2 1 2 3 1 1 2 3 2 160 3 1 2 1 3 1 1 3 2 3 2 1 2 2 1 1 3 21 1 3 2 2 1 161 2 1 2 3 1 1 2 2 1 2 3 1 3 1 1 3 1 1 2 1 3 1 3 2 162 2 22 3 2 2 1 2 3 1 1 3 2 3 1 2 2 2 3 2 2 2 3 2 163 3 2 1 1 1 3 1 2 2 3 2 32 2 1 2 1 2 3 1 1 1 2 3 164 2 2 3 2 3 1 2 1 3 2 1 3 2 2 1 3 1 2 1 2 2 23 2 165 3 1 1 2 2 1 1 3 1 2 1 1 1 3 1 1 3 1 3 1 1 3 2 1 166 3 1 2 2 3 21 3 1 1 2 3 1 1 2 2 2 3 2 1 3 2 1 2 167 1 1 1 2 1 1 3 1 3 1 3 1 3 1 1 23 1 2 2 2 1 3 2 168 1 1 2 2 1 2 3 2 3 1 1 2 1 3 1 2 2 3 2 2 3 1 1 3 1692 2 1 1 3 1 2 2 2 1 2 3 2 3 1 2 1 3 2 1 3 1 3 2 170 2 2 1 1 1 3 1 2 1 32 3 2 2 2 3 2 2 3 2 3 2 2 1 171 2 1 2 2 3 1 2 2 2 1 2 3 1 1 3 1 3 2 1 21 3 2 3 172 1 1 1 2 2 2 3 1 2 3 1 3 2 1 3 2 2 2 1 1 3 1 3 1 173 1 2 1 11 3 2 2 3 2 2 2 3 1 2 3 2 2 2 3 1 1 2 3 174 3 1 2 2 3 2 3 1 2 3 1 1 2 11 2 3 2 2 1 2 2 3 1 175 3 1 2 3 1 1 3 1 1 1 2 1 2 3 1 2 1 2 3 1 1 2 1 3176 2 2 1 1 1 3 2 2 1 2 2 3 1 1 3 2 3 1 1 3 2 2 3 1 177 2 2 3 2 1 1 3 11 1 2 1 3 1 3 1 2 2 2 3 2 3 2 2 178 3 1 3 1 2 2 3 1 3 2 2 2 1 1 3 2 1 22 1 3 1 2 2 179 1 3 2 3 1 2 1 1 2 1 3 1 1 2 3 1 2 1 1 1 2 3 2 3 180 3 12 1 1 2 1 3 2 3 1 1 2 2 2 3 1 3 2 2 3 2 1 2 181 1 3 1 2 1 2 2 2 3 2 1 32 1 3 1 1 1 3 2 1 2 3 2 182 3 2 2 1 2 3 1 1 2 3 2 2 3 1 1 2 2 2 3 1 1 23 2 183 1 2 3 1 1 1 3 1 2 2 2 1 3 2 2 3 2 3 1 3 1 2 1 2 184 1 1 1 2 1 31 3 1 1 3 2 2 1 2 3 1 2 3 2 3 1 2 1 185 2 2 1 3 2 3 1 3 1 1 1 2 3 2 2 21 1 2 3 2 3 1 2 186 2 3 1 1 3 1 1 2 1 2 3 2 3 1 1 1 2 2 1 3 2 2 2 3 1873 2 2 2 3 1 2 1 3 2 2 2 1 1 2 3 1 3 2 1 2 2 3 1 188 3 2 2 3 2 1 1 3 2 11 2 3 1 2 1 1 1 3 2 1 2 3 1 189 2 1 1 3 1 3 2 1 3 2 1 1 2 2 3 2 2 3 2 22 1 3 1 190 2 2 2 3 1 3 1 3 1 3 2 1 2 3 2 1 2 3 1 2 2 1 2 2 191 1 2 2 31 2 2 3 2 3 1 1 2 2 1 3 1 2 1 3 1 1 3 1 192 3 1 2 2 1 3 2 1 2 2 2 1 3 21 3 2 1 1 2 1 3 1 3 193 2 1 2 3 2 1 2 2 1 3 1 3 1 2 1 2 2 3 1 1 1 3 2 3194 2 1 2 3 2 3 1 1 1 3 2 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1 3 2 1 2 32 3 2 3 2 1 2 3 1 2 1 2 2 2 1014 1 1 1 3 1 2 1 1 3 1 3 2 2 1 3 2 1 1 1 22 3 2 3 1015 1 1 3 1 1 2 2 1 3 1 3 1 1 2 1 1 3 2 3 2 3 1 2 1 1016 3 1 21 1 3 1 1 1 3 2 3 1 1 1 2 3 2 1 1 1 2 2 3 1017 3 1 2 3 1 1 1 3 1 2 3 2 22 1 1 1 3 2 2 2 3 2 2 1018 1 3 2 3 2 1 1 3 2 1 1 2 1 1 3 2 2 2 3 1 3 1 11 1019 3 2 2 3 1 3 1 1 2 2 1 3 1 1 2 2 2 3 1 2 1 1 1 3 1020 2 2 1 1 3 11 1 2 2 2 3 2 1 2 3 2 3 2 2 3 2 2 3 1021 1 3 1 1 3 1 2 2 2 1 3 1 2 3 1 11 2 3 1 3 2 2 2 1022 2 1 1 3 2 2 2 3 1 3 1 2 1 1 1 3 1 2 3 1 2 1 2 31023 2 3 1 3 1 2 1 3 2 2 2 3 2 1 1 2 1 2 3 2 2 2 3 2 1024 1 3 2 2 2 3 11 1 2 2 3 2 1 1 3 2 2 2 3 1 2 3 1 1025 2 1 3 1 1 2 2 3 1 2 2 1 1 2 3 1 23 1 3 2 1 3 2 1026 1 3 1 3 1 2 2 2 3 2 1 1 2 1 1 3 2 1 2 2 3 1 1 3 10271 2 1 1 2 3 1 2 3 2 1 1 2 3 2 1 1 3 2 1 3 2 3 2 1028 2 3 1 1 1 2 2 2 3 12 3 1 3 1 3 1 2 1 2 3 2 2 1 1029 2 3 2 3 2 1 1 1 3 2 1 2 1 3 2 2 2 1 2 32 2 1 3 1030 2 3 1 1 2 1 1 3 2 3 1 1 1 2 1 3 1 1 2 3 1 1 2 3 1031 1 1 13 1 1 1 3 1 2 2 3 2 1 1 2 1 1 3 2 1 3 1 3 1032 1 1 2 3 1 1 1 2 1 3 2 3 22 1 1 1 2 3 1 3 2 3 2 1033 3 2 1 3 1 2 1 1 1 3 1 2 3 2 3 1 1 2 2 1 2 3 12 1034 3 1 2 1 3 2 1 2 1 2 3 2 3 2 3 2 1 2 2 2 3 2 2 2 1035 1 2 3 2 2 23 2 1 3 1 1 1 2 3 2 2 2 3 1 1 3 1 2 1036 1 1 1 2 2 2 3 2 1 3 1 3 1 3 1 11 2 2 2 3 2 2 3 1037 2 1 3 1 1 2 1 1 3 1 2 2 1 3 2 1 1 3 2 3 2 1 3 11038 2 3 1 2 2 2 1 3 1 3 1 1 1 2 1 2 3 1 3 2 1 3 1 1 1039 1 1 2 1 3 1 32 1 2 3 2 2 3 2 2 2 1 2 3 1 3 1 1 1040 3 1 2 3 1 2 3 1 1 3 1 3 2 2 2 1 22 3 2 1 1 1 2 1041 1 1 3 2 1 1 1 3 1 1 3 1 1 3 1 1 1 2 3 2 3 2 2 1 10422 2 3 1 1 3 1 1 2 2 1 1 3 2 3 2 2 2 1 3 2 3 2 1 1043 1 3 1 1 1 3 1 1 2 32 2 3 1 2 2 2 1 2 3 1 2 3 2 1044 3 1 2 2 1 1 1 3 1 3 1 2 3 2 2 3 1 2 2 31 1 1 2 1045 1 1 2 3 1 2 1 1 2 2 3 2 2 3 1 3 1 3 1 3 2 1 1 2 1046 3 2 22 3 2 2 3 1 1 1 3 2 3 2 1 1 1 3 2 1 2 1 2 1047 2 3 1 3 2 2 1 2 1 2 3 1 31 1 1 3 2 3 2 1 1 2 2 1048 2 2 3 2 3 1 3 1 1 1 3 1 1 3 2 1 2 1 2 1 3 1 12 1049 3 2 1 1 3 2 2 2 1 3 1 3 2 2 1 2 1 3 1 3 2 2 2 1 1050 3 1 2 1 3 12 1 3 1 2 1 1 3 2 2 1 1 2 2 3 1 1 3 1051 1 3 1 3 1 2 3 1 2 2 3 2 2 2 1 23 2 1 2 2 1 2 3 1052 1 1 1 3 2 2 1 1 3 1 1 1 2 2 3 2 1 3 2 3 1 2 1 31053 2 2 2 3 1 2 1 2 2 3 2 2 2 3 2 3 1 3 2 3 2 1 2 1 1054 1 2 2 2 3 2 13 1 1 1 3 2 2 3 2 2 1 2 3 1 3 2 2 1055 3 1 2 2 2 3 1 3 2 1 1 3 2 2 2 1 21 3 1 2 3 1 1 1056 1 1 3 1 2 1 1 1 3 2 3 1 3 2 2 3 1 2 2 2 1 3 1 2 10573 1 2 1 2 2 3 2 1 1 3 1 2 1 2 3 2 2 3 2 1 1 1 3 1058 3 2 1 1 3 1 3 2 3 21 2 2 3 2 1 1 3 2 2 1 1 2 2 1059 3 2 3 2 3 1 2 2 1 3 2 1 1 2 3 1 1 3 2 12 2 2 1 1060 3 2 1 1 3 1 1 1 3 1 2 2 1 1 3 2 3 2 2 1 3 2 1 1 1061 1 3 21 3 1 1 1 3 2 2 3 1 1 1 2 2 3 1 2 2 1 2 3 1062 2 1 1 3 1 3 1 1 3 2 2 3 13 2 1 1 2 3 2 1 2 2 2 1063 3 2 2 1 1 3 1 1 1 2 1 3 2 1 3 1 2 1 1 3 2 3 11 1064 2 1 1 3 2 1 1 1 2 2 3 1 1 1 3 2 3 2 1 2 1 3 2 3 1065 1 1 3 1 2 32 1 2 3 2 2 2 1 2 2 3 2 2 3 2 3 2 1 1066 1 2 2 2 1 3 1 1 2 1 2 1 3 2 3 11 3 1 3 1 2 1 3 1067 3 2 2 1 2 3 1 1 1 3 1 3 2 1 2 3 2 3 2 2 1 1 1 21068 2 1 2 2 1 2 3 2 3 1 1 3 1 1 3 1 1 2 3 1 2 2 1 3 1069 2 1 1 2 1 1 32 2 3 1 1 3 1 3 1 1 2 2 3 2 2 3 2 1070 2 3 1 2 3 2 2 2 3 1 2 3 2 1 1 2 23 2 2 1 1 1 3 1071 3 2 3 1 1 1 3 1 2 2 2 3 1 3 2 2 2 3 2 1 2 1 1 2 10721 3 1 3 1 1 2 1 2 1 3 1 2 2 3 1 3 1 2 2 2 3 2 2 1073 2 2 2 3 1 3 1 2 3 23 1 2 3 1 2 1 1 1 3 2 2 1 1 1074 3 2 2 3 2 1 1 1 2 2 3 2 1 3 2 1 1 1 3 11 3 2 1 1075 3 2 3 2 2 1 2 3 1 2 3 2 2 3 2 2 2 3 2 1 2 2 1 2 1076 1 2 21 2 2 3 2 3 2 1 3 1 2 3 2 1 2 2 1 1 3 1 3 1077 3 2 2 1 3 1 1 1 3 1 2 2 21 3 1 1 3 2 2 1 3 2 2 1078 2 2 3 2 3 2 1 2 2 1 1 3 1 3 1 3 2 3 1 1 1 2 12 1079 3 2 2 2 1 1 3 1 2 1 3 1 1 1 3 1 3 2 3 1 2 2 2 1 1080 1 1 2 3 1 31 1 1 2 1 3 1 2 1 3 2 2 1 2 2 3 2 3 1081 2 3 1 1 2 2 3 1 1 2 1 1 3 1 1 22 2 3 2 2 3 2 3 1082 1 1 2 1 1 3 1 2 2 3 1 1 2 2 1 3 2 3 1 3 2 1 1 31083 1 1 2 3 2 2 2 3 1 3 1 3 1 2 2 2 1 3 2 1 1 1 3 1 1084 1 3 2 2 2 1 31 1 2 1 3 1 1 1 2 3 2 3 2 2 2 3 1 1085 2 1 2 1 1 3 2 1 1 3 2 3 2 2 1 1 31 2 2 2 3 1 3 1086 3 2 1 3 2 3 1 1 2 1 1 3 2 2 1 3 2 3 2 2 1 1 2 1 10871 1 3 2 3 2 3 2 2 1 1 1 3 2 1 1 1 2 3 2 1 3 1 2 1088 1 3 1 3 1 2 3 2 2 21 2 3 2 2 3 2 3 1 1 2 2 1 1 1089 1 3 2 2 3 1 1 2 1 2 2 3 1 2 3 1 2 1 1 31 1 3 1 1090 2 3 1 1 2 3 2 3 1 3 1 2 3 2 2 2 1 3 1 1 2 1 1 2 1091 1 1 21 1 2 3 1 2 3 2 1 1 3 2 2 2 3 1 3 2 2 2 3 1092 1 1 1 3 1 3 2 3 1 1 2 1 31 1 1 2 1 1 3 1 3 1 1 1093 1 1 2 1 1 1 3 2 2 1 2 2 3 1 3 1 3 1 3 2 2 2 13 1094 1 3 2 1 3 2 3 2 2 3 2 1 3 2 2 2 1 3 2 1 2 1 2 1 1095 3 2 1 1 3 11 2 3 2 1 2 2 1 3 1 2 1 2 2 2 3 2 3 1096 3 1 2 1 1 1 2 3 2 2 2 3 1 2 1 11 3 2 1 3 2 2 3 1097 1 2 1 3 2 1 2 3 2 1 2 3 2 3 2 3 1 1 3 1 2 2 2 11098 1 2 3 1 1 2 3 2 1 3 1 3 2 3 1 2 2 1 3 2 2 2 1 1 1099 3 2 1 3 2 1 22 2 1 3 2 3 1 2 3 2 1 1 3 1 1 2 1 1100 1 3 1 1 2 2 3 2 1 2 2 3 1 1 3 1 13 1 1 2 1 2 3 1101 2 2 2 1 2 1 3 1 1 2 2 3 1 3 1 3 1 1 3 2 2 1 1 3 11021 1 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 1 3 1 1 2 2 1 1103 2 2 2 1 1 1 3 1 1 13 2 1 2 2 3 2 1 1 3 1 3 2 3 1104 1 1 1 2 2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 13 2 2 2 1105 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1106 1 2 22 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1107 1 1 3 1 3 1 1 2 1 1 2 3 21 3 1 3 1 2 1 2 1 1 3 1108 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 11 1109 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1110 1 2 2 3 2 32 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 1111 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 31 2 2 3 1 2 3 2 1112 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 11113 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 1114 2 2 1 1 1 2 31 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 1115 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 12 3 1 1 2 1 1 1116 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 11172 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 1118 2 2 2 3 1 1 2 3 1 11 2 2 3 1 2 3 1 2 1 3 1 2 3 1119 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 31 1 1 3 1120 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 1121 2 2 21 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 1122 2 1 1 1 3 2 3 1 1 1 3 1 22 2 3 1 1 1 2 3 1 2 3 1123 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 22 1124 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 1125 2 2 3 2 3 11 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 1126 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 22 3 2 3 2 2 3 1 1127 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 21128 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 1129 2 2 3 2 1 2 22 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 1130 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 13 1 1 1 2 1 2 1131 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 11322 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 1133 3 2 2 3 2 1 1 2 2 23 1 3 2 3 2 2 1 3 2 2 1 2 2 1134 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 22 3 2 3 1135 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 1136 3 1 23 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1137 1 3 2 3 1 3 1 2 2 2 1 3 11 3 1 2 3 2 2 1 2 2 1 1138 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 32 1139 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 1140 2 1 3 1 3 22 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 1141 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 13 1 1 3 1 2 2 2 1142 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 21143 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 1144 3 1 2 3 1 1 31 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 1145 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 12 2 3 1 3 2 1 1146 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 11473 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1148 1 2 2 2 3 1 3 2 1 22 2 3 2 3 2 1 2 2 3 1 1 2 3 1149 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 12 2 3 2 1150 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1151 1 2 21 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1152 1 1 3 1 1 2 1 3 1 2 3 1 31 2 2 1 3 1 1 1 2 1 3 1153 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 13 1154 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1155 1 3 1 3 1 11 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 1156 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 32 1 2 3 1 1 2 1 1157 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 31158 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 1159 2 2 3 1 2 2 23 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1160 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 31 1 2 2 1 3 1 1161 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 11622 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1163 1 2 2 3 1 1 2 2 3 12 1 2 1 3 2 3 2 1 1 1 3 2 3 1164 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 31 3 2 2 1165 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1166 1 1 21 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 1167 3 2 1 3 1 3 1 2 1 1 2 2 31 2 3 2 3 2 1 1 2 2 2 1168

In Table IA, each of the numerals 1 to 3 (numeric identifiers)represents a nucleotide base and the pattern of numerals 1 to 3 of thesequences in the above list corresponds to the pattern of nucleotidebases present in the oligonucleotides of Table I, which oligonucleotideshave been found to be non-cross-hybridizing, as described further in thedetailed examples. Each nucleotide base is selected from the group ofnucleotide bases consisting of A, C, G, and T/U. A particularlypreferred embodiment of the invention, in which a specific base isassigned to each numeric identifier is shown in Table I, below.

In one broad aspect, the invention is a composition comprising moleculesfor use as tags or tag complements wherein each molecule comprises anoligonucleotide selected from a set of oligonucleotides based on a groupof sequences as specified by numeric identifiers set out in Table IA. Inthe sequences, each of 1 to 3 is a nucleotide base selected to bedifferent from the others of 1 to 3 with the proviso that up to threenucleotide bases of each sequence can be substituted with any nucleotidebase provided that:

for any pair of sequences of the set:

-   -   M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19, where:        -   M1 is the maximum number of matches for any alignment in            which there are no internal indels;        -   M2 is the maximum length of a block of matches for any            alignment;        -   M3 is the maximum number of matches for any alignment having            a maximum score;        -   M4 is the maximum sum of the lengths of the longest two            blocks of matches for any alignment of maximum score; and        -   M5 is the maximum sum of the lengths of all the blocks of            matches having a length of at least 3, for any alignment of            maximum score; wherein:            -   the score of an alignment is determined according to the                equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:                -   for each of (i) to (iv):                -    (i) m=6, mm=6, og=0 and eg=6,                -    (ii) m=6, mm=6, og=5 and eg=1,                -    (iii) m=6, mm=2, og=5 and eg=1, and                -    (iv) m=6, mm=6, og=6 and eg=0,                -   A is the total number of matched pairs of bases in                    the alignment;                -   B is the total number of internal mismatched pairs                    in the alignment;                -   C is the total number of internal gaps in the                    alignment; and                -   D is the total number of internal indels in the                    alignment minus the total number of internal gaps in                    the alignment; and        -   wherein the maximum score is determined separately for each            of (i), (ii), (iii) and (iv).

An explanation of the meaning of the parameters set out above is givenin the section describing detailed embodiments.

In another broad aspect, the invention is a composition containingmolecules for use as tags or tag complements wherein each moleculecomprises an oligonucleotide selected from a set of oligonucleotidesbased on a group of sequences as set out in Table IA wherein each of 1to 3 is a nucleotide base selected to be different from the others of 1to 3 with the proviso that up to three nucleotide bases of each sequencecan be substituted with any nucleotide base provided that:

for any pair of sequences of the set:

-   -   M1≦19, M2≦17, M3≦21, M4≦18, and M5≦20, where:        -   M1 is the maximum number of matches for any alignment in            which there are no internal indels;        -   M2 is the maximum length of a block of matches for any            alignment;        -   M3 is the maximum number of matches for any alignment having            a maximum score;        -   M4 is the maximum sum of the lengths of the longest two            blocks of matches for any alignment of maximum score; and        -   M5 is the maximum sum of the lengths of all the blocks of            matches having a length of at least 3, for any alignment of            maximum score; wherein            -   the score of an alignment is determined according to the                equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:                -   for each of (i) to (iv):                -    (i) m=6, mm=6, og=0 and eg=6,                -    (ii) m=6, mm=6, og=5 and eg=1,                -    (iii) m=6, mm=2, og=5 and eg=1, and                -    (iv) m=6, mm=6, og=6 and eg=0,                -   A is the total number of matched pairs of bases in                    the alignment;                -   B is the total number of internal mismatched pairs                    in the alignment;                -   C is the total number of internal gaps in the                    alignment; and                -   D is the total number of internal indels in the                    alignment minus the total number of internal gaps in                    the alignment; and        -   wherein the maximum score is determined separately for each            of (i), (ii), (iii) and (iv).

In another broad aspect, the invention is a composition comprisingmolecules for use as tags or tag complements wherein each moleculecomprises an oligonucleotide selected from a set of oligonucleotidesbased on a group of sequences set out in Table IA wherein each of 1 to 3is a nucleotide base selected to be different from the others of 1 to 3with the proviso that up to three nucleotide bases of each sequence canbe substituted with any nucleotide base provided that:

for any pair of sequences of the set:

-   -   M1≦19, M2≦17, M3≦21, M4≦18, and M5≦20, where:        -   M1 is the maximum number of matches for any alignment in            which there are no internal indels;        -   M2 is the maximum length of a block of matches for any            alignment;        -   M3 is the maximum number of matches for any alignment having            a maximum score;        -   M4 is the maximum sum of the lengths of the longest two            blocks of matches for any alignment of maximum score; and        -   M5 is the maximum sum of the lengths of all the blocks of            matches having a length of at least 3, for any alignment of            maximum score, wherein:            -   the score of an alignment is determined according to the                equation 3A−B−3C−D, wherein:                -   A is the total number of matched pairs of bases in                    the alignment;                -   B is the total number of internal mismatched pairs                    in the alignment;                -   C is the total number of internal gaps in the                    alignment; and                -   D is the total number of internal indels in the                    alignment minus the total number of internal gaps in                    the alignment.

In preferred aspects, the invention provides a composition in which, forthe group of 24mer sequences in which 1=A, 2=T and 3=G, under a definedset of conditions in which the maximum degree of hybridization between asequence and any complement of a different sequence of the group of24mer sequences does not exceed 30% of the degree of hybridizationbetween said sequence and its complement, for all said oligonucleotidesof the composition, the maximum degree of hybridization between anoligonucleotide and a complement of any other oligonucleotide of thecomposition does not exceed 50% of the degree of hybridization of theoligonucleotide and its complement.

More preferably, the maximum degree of hybridization between a sequenceand any complement of a different sequence does not exceed 30% of thedegree of hybridization between said sequence and its complement, thedegree of hybridization between each sequence and its complement variesby a factor of between 1 and up to 10, more preferably between 1 and upto 9, more preferably between 1 and up to 8, more preferably between 1and up to 7, more preferably between 1 and up to 6, and more preferablybetween 1 and up to 5.

It is also preferred that the maximum degree of hybridization between asequence and any complement of a different sequence does not exceed 25%,more preferably does not exceed 20%, more preferably does not exceed15%, more preferably does not exceed 10%, more preferably does notexceed 5%.

Even more preferably, the above-referenced defined set of conditionsresults in a level of hybridization that is the same as the level ofhybridization obtained when hybridization conditions include 0.2 M NaCl,0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37° C.

In the composition, the defined set of conditions can include the groupof 24mer sequences being covalently linked to beads.

In a particular preferred aspect, for the group of 24mers the maximumdegree of hybridization between a sequence and any complement of adifferent sequence does not exceed 15% of the degree of hybridizationbetween said sequence and its complement and the degree of hybridizationbetween each sequence and its complement varies by a factor of between 1and up to 9, and for all oligonucleotides of the set, the maximum degreeof hybridization between an oligonucleotide and a complement of anyother oligonucleotide of the set does not exceed 20% of the degree ofhybridization of the oligonucleotide and its complement.

It is possible that each 1 is one of A, T/U, G and C; each 2 is one ofA, T/U, G and C; and each 3 is one of A, T/U, G and C; and each of 1, 2and 3 is selected so as to be different from all of the others of 1, 2and 3. More preferably, 1 is A or T/U, 2 is A or T/U and 3 is G or C.Even more preferably, 1 is A, 2 is T/U, and 3 is G.

In certain preferred composition, each of the oligonucleotides is fromtwenty-two to twenty-six bases in length, or from twenty-three totwenty-five, and preferably, each oligonucleotide is of the same lengthas every other said oligonucleotide.

In a particularly preferred embodiment, each oligonucleotide istwenty-four bases in length.

It is preferred that no oligonucleotide contains more than fourcontiguous bases that are identical to each other.

It is also preferred that the number of G's in each oligonucleotide doesnot exceed L/4 where L is the number of bases in said sequence.

For reasons described below, the number of G's in each saidoligonucleotide is preferred not to vary from the average number of G'sin all of the oligonucleotides by more than one. Even more preferably,the number of G's in each said oligonucleotide is the same as everyother said oligonucleotide. In the embodiment disclosed below in whicholigonucleotides were tested, the sequence of each was twenty-four basesin length and each oligonucleotide contained 6 G's.

It is also preferred that, for each nucleotide, there is at most sixbases other than G between every pair of neighboring pairs of G's.

Also, it is preferred that, at the 5′-end of each oligonucleotide atleast one of the first, second, third, fourth, fifth, sixth and seventhbases of the sequence of the oligonculeotide is a G. Similarly, it ispreferred, at the 3′-end of each oligonucleotide that at least one ofthe first, second, third, fourth, fifth, sixth and seventh bases of thesequence of the oligonucleotide is a G.

It is possible to have sequence compositions that include one hundredand sixty said molecules, or that include one hundred and seventy saidmolecules, or that include one hundred and eighty said molecules, orthat include one hundred and ninety said molecules, or that include twohundred said molecules, or that include two hundred and twenty saidmolecules, or that include two hundred and forty said molecules, or thatinclude two hundred and sixty said molecules, or that include twohundred and eighty said molecules, or that include three hundred saidmolecules, or that include four hundred said molecules, or that includefive hundred said molecules, or that include six hundred said molecules,or that include seven hundred said molecules, or that include eighthundred said molecules, or that include nine hundred said molecules, orthat include one thousand said molecules.

It is possible, in certain applications, for each molecule to be linkedto a solid phase support so as to be distinguishable from a mixturecontaining other of the molecules by hybridization to its complement.Such a molecule can be linked to a defined location on a solid phasesupport such that the defined location for each molecule is differentthan the defined location for different others of the molecules.

In certain embodiments, each solid phase support is a microparticle andeach said molecule is covalently linked to a different microparticlethan each other different said molecule.

In another broad aspect, the invention is a composition comprising a setof 150 molecules for use as tags or tag complements wherein eachmolecule includes an oligonucleotide having a sequence of at leastsixteen nucleotide bases wherein for any pair of sequences of the set:

-   -   M1≦9/24×L1, M2≦17/24×L1, M3≦21/24×L1, M4≦18/24×L1, M5≦20/24×L1,        where L1 is the length of the shortest sequence of the pair,        where:        -   M1 is the maximum number of matches for any alignment of the            pair of sequences in which there are no internal indels;        -   M2 is the maximum length of a block of matches for any            alignment of the pair of sequences;        -   M3 is the maximum number of matches for any alignment of the            pair of sequences having a maximum score;        -   M4 is the maximum sum of the lengths of the longest two            blocks of matches for any alignment of the pair of sequences            of maximum score; and        -   M5 is the maximum sum of the lengths of all the blocks of            matches having a length of at least 3, for any alignment of            the pair of sequences of maximum score, wherein:            -   the score of an alignment is determined according to the                equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:                -   for each of (i) to (iv):                -    (i) m=6, mm=6, og=0 and eg=6,                -    (ii) m=6, mm=6, og=5 and eg=1,                -    (iii) m=6, mm=2, og=5 and eg=1, and                -    (iv) m=6, mm=6, og=6 and eg=0,                -   A is the total number of matched pairs of bases in                    the alignment;                -   B is the total number of internal mismatched pairs                    in the alignment;                -   C is the total number of internal gaps in the                    alignment; and                -   D is the total number of internal indels in the                    alignment minus the total number of internal gaps in                    the alignment; and        -   wherein the maximum score is determined separately for each            of (i), (ii), (iii) and (iv).

In yet another broad aspect, the invention is a composition thatincludes a set of 150 molecules for use as tags or tag complementswherein each molecule has an oligonucleotide having a sequence of atleast sixteen nucleotide bases wherein for any pair of sequences of theset:

-   -   M1≦19, M2≦17, M3≦21, M4≦18, and M5≦0, where:        -   M1 is the maximum number of matches for any alignment of the            pair of sequences in which there are no internal indels;        -   M2 is the maximum length of a block of matches for any            alignment of the pair of sequences;        -   M3 is the maximum number of matches for any alignment of the            pair of sequences having a maximum score;        -   M4 is the maximum sum of the lengths of the longest two            blocks of matches for any alignment of the pair of sequences            of maximum score; and        -   M5 is the maximum sum of the lengths of all the blocks of            matches having a length of at least 3, for any alignment of            the pair of sequences of maximum score, wherein:            -   the score of a said alignment is determined according to                the equation 3A−B−3C−D, wherein:                -   A is the total number of matched pairs of bases in                    the alignment;                -   B is the total number of internal mismatched pairs                    in the alignment;                -   C is the total number of internal gaps in the                    alignment; and                -   D is the total number of internal indels in the                    alignment minus the total number of internal gaps in                    the alignment.

In certain embodiments of the invention, each sequence of a compositionhas up to fifty bases. More preferably, however, each sequence isbetween sixteen and forty bases in length, or between sixteen andthirty-five bases in length, or between eighteen and thirty bases inlength, or between twenty and twenty-eight bases in length, or betweentwenty-one and twenty-seven bases in length, or between twenty-two andtwenty-six bases in length.

Often, each sequence is of the same length as every other said sequence.In particular embodiments disclosed herein, each sequence is twenty-fourbases in length.

Again, it can be preferred that no sequence contains more than fourcontiguous bases that are identical to each other, etc., as describedabove.

In certain preferred embodiments, the composition is such that, under adefined set of conditions, the maximum degree of hybridization betweenan oligonucleotide and any complement of a different oligonucleotide ofthe composition does not exceed about 30% of the degree of hybridizationbetween said oligonucleotide and its complement, more preferably 20%,more preferably 15%, more preferably 10%, more preferably 6%.

Preferably, the set of conditions results in a level of hybridizationthat is the same as the level of hybridization obtained whenhybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% TritonX-100, pH 8.0 at 37° C., and the oligonucleotides are covalently linkedto microparticles. Of course it is possible that these specificconditions be used for determining the level of hybridization.

It is also preferred that under such a defined set of conditions, thedegree of hybridization between each oligonucleotide and its complementvaries by a factor of between 1 and up to 8, more preferably up to 7,more preferably up to 6, more preferably up to 5. In a particulardisclosed embodiment, the observed variance in the degree ofhybridization was a factor of only 5.3, i.e., the degree ofhybridization between each oligonucleotide and its complement varied bya factor of between 1 and 5.6.

In certain preferred embodiments, under the defined set of conditions,the maximum degree of hybridization between a said oligonucleotide andany complement of a different oligonucleotide of the composition doesnot exceed about 15%, more preferably 10%, more preferably 6%.

In one preferred embodiment, the set of conditions results in a level ofhybridization that is the same as the level of hybridization obtainedwhen hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08%Triton X-100, pH 8.0 at 37° C., and the oligonucleotides are covalentlylinked to microparticles.

Also, under the defined set of conditions, it is preferred that thedegree of hybridization between each oligonucleotide and its complementvaries by a factor of between 1 and up to 8, more preferably up to 7,more preferably up to 6, more preferably up to 5.

Any composition of the invention can include one hundred and sixty ofthe oligonucleotide molecules, or one hundred and seventy of theoligonucleotide molecules, or one hundred and eighty of theoligonucleotide molecules, or one hundred and ninety of theoligonucleotide molecules, or two hundred of the oligonucleotidemolecules, or two hundred and twenty of the oligonucleotide molecules,or two hundred and forty of the oligonucleotide molecules, or twohundred and sixty of the oligonucleotide molecules, or two hundred andeighty of the oligonucleotide molecules, or three hundred of theoligonucleotide molecules, or four hundred of the oligonucleotidemolecules, or five hundred of the oligonucleotide molecules, or sixhundred of the oligonucleotide molecules, or seven hundred of theoligonucleotide molecules, or eight hundred of the oligonucleotidemolecules, or nine hundred of the oligonucleotide molecules, or onethousand or more of the oligonucleotide molecules.

A composition of the invention can be a family of tags, or it can be afamily of tag complements.

An oligonucleotide molecule belonging to a family of molecules of theinvention can have incorporated thereinto one more analogues ofnucleotide bases, preference being given those that undergo normalWatson-Crick base pairing.

The invention includes kits for sorting and identifying polynucleotides.Such a kit can include one or more solid phase supports each having oneor more spatially discrete regions, each such region having a uniformpopulation of substantially identical tag complements covalentlyattached. The tag complements are made up of a set of oligonucleotidesof the invention.

The one or more solid phase supports can be a planar substrate in whichthe one or more spatially discrete regions is a plurality of spatiallyaddressable regions.

The tag complements can also be coupled to microparticles.Microparticles preferably each have a diameter in the range of from 5 to40 μm.

Such a kit preferably includes microparticles that arespectrophotometrically unique, and therefore distinguisable from eachother according to conventional laboratory techniques. Of course forsuch kits to work, each type of microparticle would generally have onlyone tag complement associated with it, and usually there would be adifferent oligonucleotide tag complement associated with (attached to)each type of microparticle.

The invention includes methods of using families of oligonucleotides ofthe invention.

One such method is of analyzing a biological sample containing abiological sequence for the presence of a mutation or polymorphism at alocus of the nucleic acid. The method includes:

-   (A) amplifying the nucleic acid molecule in the presence of a first    primer having a 5′-sequence having the sequence of a tag    complementary to the sequence of a tag complement belonging to a    family of tag complements of the invention to form an amplified    molecule with a 5′-end with a sequence complementary to the sequence    of the tag;-   (B) extending the amplified molecule in the presence of a polymerase    and a second primer having 5′-end complementary the 3′-end of the    amplified sequence, with the 3′-end of the second primer extending    to immediately adjacent said locus, in the presence of a plurality    of nucleoside triphosphate derivatives each of which is: (i) capable    of incorporation during transciption by the polymerase onto the    3′-end of a growing nucleotide strand; (ii) causes termination of    polymerization; and (iii) capable of differential detection, one    from the other, wherein there is a said derivative complementary to    each possible nucleotide present at said locus of the amplified    sequence;-   (C) specifically hybridizing the second primer to a tag complement    having the tag complement sequence of (A); and-   (B) detecting the nucleotide derivative incorporated into the second    primer in (B) so as to identify the base located at the locus of the    nucleic acid.

In another method of the invention, a biological sample containing aplurality of nucleic acid molecules is analyzed for the presence of amutation or polymorphism at a locus of each nucleic acid molecule, foreach nucleic acid molecule. This method includes steps of:

-   (A) amplifying the nucleic acid molecule in the presence of a first    primer having a 5′-sequence having the sequence of a tag    complementary to the sequence of a tag complement belonging to a    family of tag complements of the invention to form an amplified    molecule with a 5′-end with a sequence complementary to the sequence    of the tag;-   (B) extending the amplified molecule in the presence of a polymerase    and a second primer having 5′-end complementary the 3′-end of the    amplified sequence, the 3′-end of the second primer extending to    immediately adjacent said locus, in the presence of a plurality of    nucleoside triphosphate derivatives each of which is: (i) capable of    incorporation during transciption by the polymerase onto the 3′-end    of a growing nucleotide strand; (ii) causes termination of    polymerization; and (iii) capable of differential detection, one    from the other, wherein there is a said derivative complementary to    each possible nucleotide present at said locus of the amplified    molecule;-   (C) specifically hybridizing the second primer to a tag complement    having the tag complement sequence of (A); and-   (D) detecting the nucleotide derivative incorporated into the second    primer in (B) so as to identify the base located at the locus of the    nucleic acid;    wherein each tag of (A) is unique for each nucleic acid molecule and    steps (A) and (B) are carried out with said nucleic molecules in the    presence of each other.

Another method includes analyzing a biological sample that contains aplurality of double stranded complementary nucleic acid molecules forthe presence of a mutation or polymorphism at a locus of each nucleicacid molecule, for each nucleic acid molecule. The method includes stepsof:

-   (A) amplifying the double stranded molecule in the presence of a    pair of first primers, each primer having an identical 5′-sequence    having the sequence of a tag complementary to the sequence of a tag    complement belonging to a family of tag complements of the invention    to form amplified molecules with 5′-ends with a sequence    complementary to the sequence of the tag;-   (B) extending the amplified molecules in the presence of a    polymerase and a pair of second primers each second primer having a    5′-end complementary a 3′-end of the amplified sequence, the 3′-end    of each said second primer extending to immediately adjacent said    locus, in the presence of a plurality of nucleoside triphosphate    derivatives each of which is: (i) capable of incorporation during    transciption by the polymerase onto the 3′-end of a growing    nucleotide strand; (ii) causes termination of polymerization;    and (iii) capable of differential detection, one from the other;-   (C) specifically hybridizing each of the second primers to a tag    complement having the tag complement sequence of (A); and-   (D) detecting the nucleotide derivative incorporated into the second    primers in (B) so as to identify the base located at said locus;    wherein the sequence of each tag of (A) is unique for each nucleic    acid molecule and steps (A) and (B) are carried out with said    nucleic molecules in the presence of each other.

In yet another aspect, the invention is a method of analyzing abiological sample containing a plurality of nucleic acid molecules forthe presence of a mutation or polymorphism at a locus of each nucleicacid molecule, for each nucleic acid molecule, the method includingsteps of:

-   (a) hybridizing the molecule and a primer, the primer having a    5′-sequence having the sequence of a tag complementary to the    sequence of a tag complement belonging to a family of tag    complements of the invention and a 3′-end extending to immediately    adjacent the locus;-   (b) enzymatically extending the 3′-end of the primer in the presence    of a plurality of nucleoside triphosphate derivatives each of which    is: (i) capable of enzymatic incorporation onto the 3′-end of a    growing nucleotide strand; (ii) causes termination of said    extension; and (iii) capable of differential detection, one from the    other, wherein there is a said derivative complementary to each    possible nucleotide present at said locus;-   (c) specifically hybridizing the extended primer formed in step (b)    to a tag complement having the tag complement sequence of (a); and-   (d) detecting the nucleotide derivative incorporated into the primer    in step (b) so as to identify the base located at the locus of the    nucleic acid molecule;    wherein each tag of (a) is unique for each nucleic acid molecule and    steps (a) and (b) are carried out with said nucleic molecules in the    presence of each other.

The derivative can be a dideoxy nucleoside triphosphate.

Each respective complement can be attached as a uniform population ofsubstantially identical complements in specially discrete regions on oneor more solid phase support(s).

Each tag complement can include a label, each such label being differentfor respective complements, and step (d) can include detecting thepresence of the different labels for respective hybridization complexesof bound tags and tag complements.

Another aspect of the invention includes a method of determining thepresence of a target suspected of being contained in a mixture. Themethod includes the steps of:

-   (i) labelling the target with a first label;-   (ii) providing a first detection moiety capable of specific binding    to the target and including a first tag;-   (iii) exposing a sample of the mixture to the detection moiety under    conditions suitable to permit (or cause) said specific binding of    the molecule and target;-   (iv) providing a family of suitable tag complements of the invention    wherein the family contains a first tag complement having a sequence    complementary to that of the first tag;-   (v) exposing the sample to the family of tag complements under    conditions suitable to permit (or cause) specific hybridization of    the first tag and its tag complement;-   (vi) determining whether a said first detection moiety hybridized to    a first said tag complement is bound to a said labelled target in    order to determine the presence or absence of said target in the    mixture.

Preferably, the first tag complement is linked to a solid support at aspecific location of the support and step (vi) includes detecting thepresence of the first label at said specified location.

Also, the first tag complement can include a second label and step (vi)includes detecting the presence of the first and second labels in ahybridized complex of the moiety and the first tag complement.

Further, the target can be selected from the group consisting of organicmolecules, antigens, proteins, polypeptides, antibodies and nucleicacids. The target can be an antigen and the first molecule can be anantibody specific for that antigen.

The antigen is usually a polypeptide or protein and the labelling stepcan include conjugation of fluorescent molecules, digoxigenin,biotinylation and the like.

The target can be a nucleic acid and the labelling step can includeincorporation of fluorescent molecules, radiolabelled nucleotide,digoxigenin, biotinylation and the like.

DETAILED DESCRIPTION OF THE INVENTION Figures

Reference is made to the attached figures in which,

FIG. 1 illustrates generally the steps followed to obtain a family ofsequences of the present invention;

FIG. 2 shows the intensity of the signal (MFI) for each perfectlymatched sequence (probe sequences indicated in Table I) and itscomplement (target, at 50 fmol) obtained as described in Example 1;

FIG. 3 is a three dimensional representation showing cross-hybridizationobserved for the sequences of FIG. 2 as described in Example 1. Theresults shown in FIG. 2 are reproduced along the diagonal of thedrawing; and

FIG. 4 is illustrative of results obtained for an individual target (SEQID NO:90, target No. 90) when exposed to the 100 probes of Example 1.The MFI for each bead is plotted.

DETAILED EMBODIMENTS

The invention provides a method for sorting complex mixtures ofmolecules by the use of families of oligonucleotide sequence tags. Thefamilies of oligonucleotide sequence tags are designed so as to provideminimal cross hybridization during the sorting process. Thus anysequence within a family of sequences will not significantlycross-hybridize with any other sequence derived from that family underappropriate hybridization conditions known by those skilled in the art.The invention is particularly useful in highly parallel processing ofanalytes.

Families of Oligonucleotide Sequence Tags

The present invention includes a family of 24mer polynucleotides thathave been demonstrated to be minimally cross-hybridizing with eachother. This family of polynucleotides is thus useful as a family oftags, and their complements as tag complements.

In order to be considered for inclusion into the family, a sequence hadto satisfy a certain number of rules regarding its composition. Forexample, repetitive regions that present potential hybridizationproblems such as four or more of a similar base (e.g., AAAA or TTTT) orpairs of Gs were forbidden. Another rule is that each sequence containsexactly six Gs and no Cs, in order to have sequences that are more orless isothermal. Also required for a 24mer to be included is that theremust be at most six bases between every neighboring pair of Gs. Anotherway of putting this is that there are at most six non-Gs between any twoconsecutive Gs. Also, each G nearest the 5′-end (resp. 3′-end) of itsoligonucleotide (the left-hand (resp. right-hand) side as written inTable I) was required to occupy one of the first to seventh positions(counting the 5′-terminal (resp. 3′-terminal) position as the firstposition.)

The process used to design families of sequences that do not exhibitcross-hybridization behavior is illustrated generally in FIG. 1).Depending on the application for which these families of sequences willbe used, various rules are designed. A certain number of rules canspecify constraints for sequence composition (such as the ones describedin the previous paragraph). The other rules are used to judge whethertwo sequences are too similar. Based on these rules, a computer programcan derive families of sequences that exhibit minimal or nocross-hybridization behavior. The exact method used by the computerprogram is not crucial since various computer programs can derivesimilar families based on these rules. Such a program is for exampledescribed in international patent application No. PCT/CA 01/00141published under WO 01/59151 on Aug. 16, 2001. Other programs can usedifferent methods, such as the ones summarized below.

A first method of generating a maximum number of minimallycross-hybridizing polynucleotide sequences starts with any number ofnon-cross-hybridizing sequences, for example just one sequence, andincreases the family as follows. A certain number of sequences isgenerated and compared to the sequences already in the family. Thegenerated sequences that exhibit too much similarity with sequencesalready in the family are dropped. Among the “candidate sequences” thatremain, one sequence is selected and added to the family. The othercandidate sequences are then compared to the selected sequence, and theones that show too much similarity are dropped. A new sequence isselected from the remaining candidate sequences, if any, and added tothe family, and so on until there are no candidate sequences left. Atthis stage, the process can be repeated (generating a certain number ofsequences and comparing them to the sequences in the family, etc.) asoften as desired. The family obtained at the end of this method containsonly minimally cross-hybridizing sequences.

A second method of generating a maximum number of minimallycross-hybridizing polynucleotide sequences starts with a fixed-sizefamily of polynucleotide sequences. The sequences of this family can begenerated randomly or designed by some other method. Many sequences inthis family may not be compatible with each other, because they show toomuch similarity and are not minimally cross-hybridizing. Therefore, somesequences need to be replaced by new ones, with less similarity. One wayto achieve this consists of repeatedly replacing a sequence of thefamily by the best (that is, lowest similarity) sequence among a certainnumber of (for example, randomly generated) sequences that are not partof the family. This process can be repeated until the family ofsequences shows minimal similarity, hence minimal cross-hybridizing, oruntil a set number of replacements has occurred. If, at the end of theprocess, some sequences do not obey the similarity rules that have beenset, they can be taken out of the family, thus providing a somewhatsmaller family that only contains minimally cross-hybridizing sequences.Some additional rules can be added to this method in order to make itmore efficient, such as rules to determine which sequence will bereplaced.

Such methods have been used to obtain the 1168 non-cross-hybridizingtags of Table I that are the subject of this patent application.

One embodiment of the invention is a composition comprising moleculesfor use as tags or tag complements wherein each molecule comprises anoligonucleotide selected from a set of oligonucleotides based on thegroup of sequences set out in Table IA, wherein each of the numericidentifiers 1 to 3 (see the Table) is a nucleotide base selected to bedifferent from the others of 1 to 3. According to this embodiment,several different families of specific sets of oligonucleotide sequencesare described, depending upon the assignment of bases made to thenumeric identifiers 1 to 3.

The sequences contained in Table I have a mathematical relationship toeach other, described as follows.

Let S and T be two DNA sequences of lengths s and t respectively. Whilethe term “alignment” of nucleotide sequences is widely used in the fieldof biotechnology, in the context of this invention the term has aspecific meaning illustrated here. An alignment of S and T is a 2xpmatrix A (with p≧s and p≧t) such that the first (or second) row of Acontains the characters of S (or T respectively) in order, interspersedwith p-s (or p-t respectively) spaces. It assumed that no column of thealignment matrix contains two spaces, i.e., that any alignment in whicha column contains two spaces is ignored and not considered here. Thecolumns containing the same base in both rows are called matches, whilethe columns containing different bases are called mismatches. Eachcolumn of an alignment containing a space in its first row is called aninsertion and each colmun containing a space in its second row is calleda deletion while a column of the alignment containing a space in eitherrow is called an indel. Insertions and deletions within a sequence arerepresented by the character ‘-’. A gap is a continuous sequence ofspaces in one of the rows (that is neither immediately preceded norimmediately followed by another space in the same row), and the lengthof a gap is the number of spaces in that gap. An internal gap is one inwhich its first space is preceded by a base and its last space isfollowed by a base and an internal indel is an indel belonging to aninternal gap. Finally, a block is a continuous sequence of matches (thatis neither immediately preceded nor immediately followed by anothermatch), and the length of a block is the number of matches in thatblock. In order to illustrate these definitions, two sequencesS=TGATCGTAGCTACGCCGCG (of length s=19; SEQ ID NO:1169) andT=CGTACGATTGCAACGT (of length t=16; SEQ ID NO:1170) are considered.Exemplary alignment R₁ of S and T (with p=23) is:

Alignment R₁: — — — — T G A T C G T A G C T A C G C C G C G C G T A C GA T — — T — G C A A C G T — — — — 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1617 18 19 20 21 22 23

Columns 1 to 4, 9, 10, 12 and 20 to 23 are indels, columns 6, 7, 8, 11,13, 14, 16, 17 and 18 are matches, and columns 5, 15 and 19 aremismatches. Columns 9 and 10 form a gap of length 2, while columns 16 to18 form a block of length 3. Columns 9, 10 and 12 are internal indels.

A score is assigned to the alignment A of two sequences by assigningweights to each of matches, mismatches and gaps as follows:

-   -   the reward for a match m,    -   the penalty for a mismatch mm,    -   the penalty for opening a gap og,    -   the penalty for extending a gap eg.        Once these values are set, a score to each column of the        alignment is assigned according to the following rules:    -   1. assign 0 to each column preceding the first match and to each        column following the last match.    -   2. for each of the remaining columns, assign in if it is a        match, −mm if it is a mismatch, −og-eg if it is the first indel        of a gap, −eg if it is an indel but not the first indel of a        gap.        The score of the alignment A is the sum of the scores of its        columns. An alignment is said to be of maximum score if no other        alignment of the same two sequences has a higher score (with the        same values of m, mm, og and eg). A person knowledgeable in the        field will recognize this method of scoring an alignment as        scoring a local (as opposed to global) alignment with affine gap        penalties (that is, gap penalties that can distinguish between        the first indel of a gap and the other indels). It will be        appreciated that the total number of indels that open a gap is        the same as the total number of gaps and that an internal indel        is not one of those assigned a 0 in rule (1) above. It will also        be noted that foregoing rule (1) assigns a 0 for non-internal        mismatches. An internal mismatch is a mismatch that is preceded        and followed (not necessarily immediately) by a match.

As an illustration, if the values of m, mm, og and eg are set to 3, 1, 2and 1 respectively, alignment R₁ has a score of 19, determined as shownbelow:

Scoring of Alignment R₁ — — — — T G A T C G T A G C T A C G C C G C G CG T A C G A T — — T — G C A A C G T — — — — 0 0 0 0 0 3 3 3 −3 −1 3 −3 33 −1 3 3 3 0 0 0 0 0Note that for two given sequences S and T, there are numerousalignments. There are often several alignments of maximum score.

Based on these alignments, five sequence similarity measures are definedas follows. For two sequences S and T, and weights {m, mm, og, eg}:

-   -   M1 is the maximum number of matches over all alignments free of        internal indels;    -   M2 is the maximum length of a block over all alignments;    -   M3 is the maximum number of matches over all alignments of        maximum score;    -   M4 is the maximum sum of the lengths of the longest two blocks        over all alignments of maximum score;    -   M5 is the maximum sum of the lengths of all the blocks of length        at least 3, over all alignments of maximum score.        Notice that, by definition, the following inequalities between        these similarity measures are obtained: M4≦M3 and M5≦M3. Also,        in order to determine M2 it is sufficient to determine the        maximum length of a block over all alignments free of internal        indels. For two given sequences, the values of M3 to M5 can vary        depending on the values of the weights {m, mm, og, eg}, but not        M1 and M2.

For weights {3, 1, 2, 1}, the illustrated alignment is not a maximumscore alignment of the two example sequences. But for weights {6, 6, 0,6} it is; hence this alignment shows that for these two examplesequences, and weights {6, 6, 0, 6}, M2≧3, M3≧9, M4≧6 and M5≧6. In orderto determine the exact values of M1 to M5, all the necessary alignmentsneed to be considered. M1 and M2 can be found by looking at the s+t−1alignments free of internal indels, where s and t are the lengths of thetwo sequences considered. Mathematical tools known as dynamicprogramming can be implemented on a computer and used to determine M3 toM5 in a very quick way. Using a computer program to do thesecalculations, it was determined that:

-   -   with the weights {6, 6, 0, 6}, M1=8, M2=4, M3=10, M4=6 and M5=6;    -   with the weights {3, 1, 2, 1}, M1=8, M2=4, M3=10, M4=6 and M5=4.        According to the preferred embodiment of this invention, two        sequences S and T each of length 24 are too similar if at least        one of the following happens:    -   M1>6 or    -   M2>13 or    -   M3>20 or    -   M4>16 or    -   M5>19        when using either weights {6, 6, 0, 6}, or {6, 6, 5, 1}, or {6,        2, 5, 1}, or {6, 6, 6, 0}. In other words, the five similarity        measures between S and T are determined for each of the above        four sets of weights, and checked against these thresholds (for        a total of 20 tests).

The above thresholds of 16, 13, 20, 16 and 19, and the above sets ofweights, were used to obtain the sequences listed in Table I. Additionalsequences can thus be added to those of Table I as long as the abovealignment rules are obeyed for all sequences.

It is also possible to alter thresholds M1, M2, etc., while remainingwithin the scope of this invention. It is thus possible to substitute oradd sequences to those of Table I, or more generally to those of TableIA to obtain other sets of sequences that would also exhibit reasonablylow cross-hybridization. More specifically, a set of 24mer sequences inwhich there are no two sequences that are too similar, where too similaris defined as:

-   -   M1>19 or    -   M2>17 or    -   M3>21 or    -   M4>18 or    -   M5>20        when using either weights {6, 6, 0, 6}, or {6, 6, 5, 1}, or {6,        2, 5, 1}, or {6, 6, 6, 0}, would also exhibit low        cross-hybridization. Reducing any of the threshold values        provides sets of sequences with even lower cross-hybridization.        Alternatively, ‘too similar’ can also be defined as:    -   M1>19 or    -   M2>17 or    -   M3>21 or    -   M4>18 or    -   M5>20        when using either weights {3, 1, 2, 1}. Alternatively, other        combinations of weights will lead to sets of sequences with low        cross-hybridization.

Notice that using weights {6, 6, 0, 6} is equivalent to using weights{1, 1, 0, 1}, or weights {2, 2, 0, 2}, . . . (that is, for any twosequences, the values of M1 to M5 are exactly the same whether weights{6, 6, 0, 6} or {1, 1, 0, 1} or {2, 2, 0, 2} or any other multiple of{1, 1, 0, 1} is used).

When dealing with sequences of length other than 24, or sequences ofvarious lengths, the definition of similarity can be adjusted. Suchadjustments are obvious to the persons skilled in the art. For example,when comparing a sequence of length L1 with a sequence of length L2(with L1<L2), they can be considered as too similar whenM1>19/24×L1M2>17/24×L1M3>21/24×L1M4>18/24×L1M5>20/24×L1when using either weights {6, 6, 0, 6}, or {6, 6, 5, 1}, or {6, 2, 5, 1}or {6, 6, 6, 0}.

Polynucleotide sequences can be composed of a subset of natural basesmost preferably A, T and G. Sequences that are deficient in one basepossess useful characteristics, for example, in reducing potentialsecondary structure formation or reduced potential for crosshybridization with nucleic acids in nature. Also, it is preferable tohave tag sequences that behave isothermally. This can be achieved forexample by maintaining a constant base composition for all sequencessuch as six Gs and eighteen As or Ts for each sequence. Additional setsof sequences can be designed by extrapolating on the original family ofnon-cross-hybridizing sequences by simple methods known to those skilledin the art.

In order to validate the sequence set, a subset of sequences from thefamily of 1168 sequence tags was selected and characterized, in terms ofthe ability of these sequences to form specific duplex structures withtheir complementary sequences, and the potential for cross-hybridizationwithin the sequence set. See Example 1, below. The subset of 100sequences was randomly selected, and analyzed using the Luminex¹⁰⁰LabMAP™ platform. The 100 sequences were chemically immobilized onto theset of 100 different Luminex microsphere populations, such that eachspecific sequence was coupled to one spectrally distinct microspherepopulation. The pool of 100 microsphere-immobilized probes was thenhybridized with each of the 100 corresponding complementary sequences.Each sequence was examined individually for its specific hybridizationwith its complementary sequence, as well as for its non-specifichybridization with the other 99 sequences present in the reaction. Thisanalysis demonstrated the propensity of each sequence to hybridize onlyto its complement (perfect match), and not to cross-hybridizeappreciably with any of the other oligonucleotides present in thehybridization reaction.

It is within the capability of a person skilled in the art, given thefamily of sequences of Table I, to modify the sequences, or add othersequences while largely retaining the property of minimalcross-hybridization which the polynucleotides of Table I have beendemonstrated to have.

There are 1168 polynucleotide sequences given in Table I. Since all 1168of this family of polynucleotides can work with each other as aminimally cross-hybridizing set, then any plurality of polynucleotidesthat is a subset of the 1168 can also act as a minimallycross-hybridizing set of polynucleotides. An application in which, forexample, 30 molecules are to be sorted using a family of polynucleotidetags and tag complements could thus use any group of 30 sequences shownin Table I. This is not to say that some subsets may be found in apractical sense to be more preferred than others. For example, it may befound that a particular subset is more tolerant of a wider variety ofconditions under which hybridization is conducted before the degree ofcross-hybridization becomes unacceptable.

It may be desirable to use polynucleotides that are shorter in lengththan the 24 bases of those in Table I. A family of subsequences (i.e.,subframes of the sequences illustrated) based on those contained inTable I having as few as 10 bases per sequence could be chosen, so longas the subsequences are chosen to retain homological properties betweenany two of the sequences of the family important to their noncross-hybridization.

The selection of sequences using this approach would be amenable to acomputerized process. Thus for example, a string of 10 contiguous basesof the first 24mer of Table I could be selected:AAATTGTGAAAGATTGTTTGTGTA (SEQ ID NO:1).

The same string of contiguous bases from the second 24mer could then beselected and compared for similarity against the first chosen sequence:GTTAGAGTTAATTGTATTTGATGA (SEQ ID NO:2). A systematic pairwise comparisoncould then be carried out to determine if the similarity requirementsare violated. If the pair of sequences does not violate any setproperty, a 10mer subsequence can be selected from the third 24mersequence of Table I, and compared to each of the first two 10mersequences (in a pairwise fashion to determine its compatibilitytherewith, etc. In this way a family of 10mer sequences may bedeveloped.

It is within the scope of this invention, to obtain families ofsequences containing 11mer, 12mer, 13mer, 14mer, 15mer, 16mer, 17mer,18mer, 19mer, 20mer, 21mer, 22mer and 23mer sequences by analogy to thatshown for 10mer sequences.

It may be desirable to have a family of sequences in which there aresequences greater in length than the 24mer sequences shown in Table I.It is within the capability of a person skilled in the art, given thefamily of sequences shown in Table I, to obtain such a family ofsequences. One possible approach would be to insert into each sequenceat one or more locations a nucleotide, non-natural base or analogue suchthat the longer sequence should not have greater similarity than any twoof the original non-cross-hybridizing sequences of Table I and theaddition of extra bases to the tag sequences should not result in amajor change in the thermodynamic properties of the tag sequences ofthat set for example the GC content must be maintained between 10%-40%with a variance from the average of 20%. This method of inserting basescould be used to obtain, for example, a family of sequences up to 40bases long.

Given a particular family of sequences that can be used as a family oftags (or tag complements), e.g., those of Table I, a skilled person willreadily recognize variant families that work equally as well.

Again taking the sequences of Table I for example, every T could beconverted to an A and vice versa and no significant change in thecross-hybridization properties would be expected to be observed. Thiswould also be true if every G were converted to a C.

Also, all of the sequences of a family could be taken to be constructedin the 5′-3′ direction, as is the convention, or all of theconstructions of sequences could be in the opposition direction (3′-5′).

There are additional modifications that can be carried out. For example,C has not been used in the family of sequences. Substitution of C inplace of one or more G's of a particular sequence would yield a sequencethat is at least as low in homology with every other sequence of thefamily as was the particular sequence chosen for modification. It isthus possible to substitute C in place of one or more G's in any of thesequences shown in Table I. Analogously, substituting of C in place ofone or more A's is possible, or substituting C in place of one or T's ispossible.

It is preferred that the sequences of a given family are of the same, orroughly the same length. Preferably, all the sequences of a family ofsequences of this invention have a length that is within five bases ofthe base-length of the average of the family. More preferably, allsequences are within four bases of the average base-length. Even morepreferably, all or almost all sequences are within three bases of theaverage base-length of the family. Better still, all or almost allsequences have a length that is within two of the base-length of theaverage of the family, and even better still, within one of thebase-length of the average of the family.

It is also possible for a person skilled in the art to derive sets ofsequences from the family of sequences described in this specificationand remove sequences that would be expected to have undesirablehybridization properties.

Methods for Synthesis of Oligonucleotide Families

Preferably oligonucleotide sequences of the invention are synthesizeddirectly by standard phosphoramidite synthesis approaches and the like(Caruthers et al, Methods in Enzymology; 154, 287-313: 1987; Lipshutz etal, Nature Genet.; 21, 20-24: 1999; Fodor et al, Science; 251, 763-773:1991). Alternative chemistries involving non natural bases such aspeptide nucleic acids or modified nucleosides that offer advantages induplex stability may also be used (Hacia et al; Nucleic Acids Res; 27:4034-4039, 1999; Nguyen et al, Nucleic Acids Res.; 27, 1492-1498: 1999;Weiler et al, Nucleic Acids Res.; 25, 2792-2799:1997). It is alsopossible to synthesize the oligonucleotide sequences of this inventionwith alternate nucleotide backbones such as phosphorothioate orphosphoroamidate nucleotides. Methods involving synthesis through theaddition of blocks of sequence in a stepwise manner may also be employed(Lyttle et al, Biotechniques, 19: 274-280 (1995). Synthesis may becarried out directly on the substrate to be used as a solid phasesupport for the application or the oligonucleotide can be cleaved fromthe support for use in solution or coupling to a second support.

Solid Phase Supports

There are several different solid phase supports that can be used withthe invention. They include but are not limited to slides, plates,chips, membranes, beads, microparticles and the like. The solid phasesupports can also vary in the materials that they are composed ofincluding plastic, glass, silicon, nylon, polystyrene, silica gel, latexand the like. The surface of the support is coated with thecomplementary tag sequences by any conventional means of attachment.

In preferred embodiments, the family of tag complement sequences isderivatized to allow binding to a solid support. Many methods ofderivatizing a nucleic acid for binding to a solid support are known inthe art (Hermanson G., Bioconjugate Techniques; Acad. Press: 1996). Thesequence tag may be bound to a solid support through covalent ornon-covalent bonds (Iannone et al, Cytometry; 39: 131-140, 2000; Matsonet al, Anal. Biochem.; 224: 110-106, 1995; Proudnikov et al, AnalBiochem; 259: 34-41, 1998; Zammatteo et al, Analytical Biochemistry;280:143-150, 2000). The sequence tag can be conveniently derivatized forbinding to a solid support by incorporating modified nucleic acids inthe terminal 5′ or 3′ locations.

A variety of moieties useful for binding to a solid support (e.g.,biotin, antibodies, and the like), and methods for attaching them tonucleic acids, are known in the art. For example, an amine-modifiednucleic acid base (available from, eg., Glen Research) may be attachedto a solid support (for example, Covalink-NH, a polystyrene surfacegrafted with secondary amino groups, available from Nunc) through abifunctional crosslinker (e.g., bis(sulfosuccinimidyl suberate),available from Pierce). Additional spacing moieties can be added toreduce steric hindrance between the capture moiety and the surface ofthe solid support.

Attaching Tags to Analytes for Sorting

A family of oligonucleotide tag sequences can be conjugated to apopulation of analytes most preferably polynucleotide sequences inseveral different ways including but not limited to direct chemicalsynthesis, chemical coupling, ligation, amplification, and the like.Sequence tags that have been synthesized with primer sequences can beused for enzymatic extension of the primer on the target for example inPCR amplification.

Detection of Single Nucleotide Polymorphisms Using Primer Extension

There are a number of areas of genetic analysis where families ofnon-cross-hybridizing sequences can be applied including diseasediagnosis, single nucleotide polymorphism analysis, genotyping,expression analysis and the like. One such approach for geneticanalysis, referred to as the primer extension method (also known asGenetic Bit Analysis (Nikiforov et al, Nucleic Acids Res.; 22,4167-4175: 1994; Head et al Nucleic Acids Res.; 25, 5065-5071: 1997)),is an extremely accurate method for identification of the nucleotidelocated at a specific polymorphic site within genomic DNA. In standardprimer extension reactions, a portion of genomic DNA containing adefined polymorphic site is amplified by PCR using primers that flankthe polymorphic site. In order to identify which nucleotide is presentat the polymorphic site, a third primer is synthesized such that thepolymorphic position is located immediately 3′ to the primer. A primerextension reaction is set up containing the amplified DNA, the primerfor extension, up to 4 dideoxynucleoside triphosphates (each labeledwith a different fluorescent dye) and a DNA polymerase such as theKlenow subunit of DNA Polymerase 1. The use of dideoxy nucleotidesensures that a single base is added to the 3′ end of the primer, a sitecorresponding to the polymorphic site. In this way the identity of thenucleotide present at a specific polymorphic site can be determined bythe identity of the fluorescent dye-labeled nucleotide that isincorporated in each reaction. One major drawback to this approach isits low throughput. Each primer extension reaction is carried outindependently in a separate tube.

Universal sequences can be used to enhance the throughput of primerextension assay as follows. A region of genomic DNA containing multiplepolymorphic sites is amplified by PCR. Alternatively, several genomicregions containing one or more polymorphic sites each are amplifiedtogether in a multiplexed PCR reaction. The primer extension reaction iscarried out as described above except that the primers used arechimeric, each containing a unique universal tag at the 5′ end and thesequence for extension at the 3′ end. In this way, each gene-specificsequence would be associated with a specific universal sequence. Thechimeric primers would be hybridized to the amplified DNA and primerextension is carried out as described above. This would result in amixed pool of extended primers, each with a specific fluorescent dyecharacteristic of the incorporated nucleotide. Following the primerextension reaction, the mixed extension reactions are hybridized to anarray containing probes that are reverse complements of the universalsequences on the primers. This would segregate the products of a numberof primer extension reactions into discrete spots. The fluorescent dyepresent at each spot would then identify the nucleotide incorporated ateach specific location. A number of additional methods for the detectionof single nucleotide polymorphisms, including but not limited to, allelespecific polymerase chain reaction (ASPCR), allele specific primerextension (ASP) and oligonucleotide ligation assay (OLA) can beperformed by someone skilled in the art in combination with the tagsequences described herein.

Kits Using Families of Tag Sequences

The families of non cross-hybridizing sequences may be provided in kitsfor use in for example genetic analysis. Such kits include at least oneset of non-cross-hybridizing sequences in solution or on a solidsupport. Preferably the sequences are attached to microparticles and areprovided with buffers and reagents that are appropriate for theapplication. Reagents may include enzymes, nucleotides, fluorescentlabels and the like that would be required for specific applications.Instructions for correct use of the kit for a given application will beprovided.

EXAMPLES Example 1 Cross Talk Behavior of Sequence on Beads

A group of 100 sequences, randomly selected from Table I, was tested forfeasibility for use as a family of minimally cross-hybridizingoligonucleotides. The 100 sequences selected are separately indicated inTable I along with the numbers assigned to the sequences in the tests.

The tests were conducted using the Luminex LabMAP™ platform availablefrom Luminex Corporation, Austin, Tex., U.S.A. The one hundredsequences, used as probes, were synthesized as oligonucleotides byIntegrated DNA Technologies (IDT, Coralville, Iowa, U.S.A.). Each probeincluded a C₆ aminolink group coupled to the 5′-end of theoligonucleotide through a C₁₂ ethylene glycol spacer. The C₆ aminolinkmolecule is a six carbon spacer containing an amine group that can beused for attaching the oligonucleotide to a solid support. One hundredoligonucleotide targets (probe complements), the sequence of each beingthe reverse complement of the 100 probe sequences, were also synthesizedby IDT. Each target was labelled at its 5′-end with biotin. Alloligonucleotides were purified using standard desalting procedures, andwere reconstituted to a concentration of approximately 200 μM insterile, distilled water for use. Oligonucleotide concentrations weredetermined spectrophotometrically using extinction coefficients providedby the supplier.

Each probe was coupled by its amino linking group to a carboxylatedfluorescent microsphere of the LapMAP system according to the Luminex¹⁰⁰protocol. The microsphere, or bead, for each probe sequence has unique,or spectrally distinct, light absorption characteristics which permitseach probe to be distinguished from the other probes. Stock bead pelletswere dispersed by sonication and then vortexing. For each beadpopulation, five million microspheres (400 μL) were removed from thestock tube using barrier tips and added to a 1.5 mL Eppendorf tube (USAScientific). The microspheres were then centrifuged, the supernatant wasremoved, and beads were resuspended in 25 μL of 0.2 M MES(2-(N-morpholino)ethane sulfonic acid) (Sigma), pH 4.5, followed byvortexing and sonication. One nmol of each probe (in a 25 μL volume) wasadded to its corresponding bead population. A volume of 2.5 μL of EDCcross-linker (1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (Pierce), prepared immediately before use by adding 1.0 mLof sterile ddH₂O to 10 mg of EDC powder, was added to each microspherepopulation. Bead mixes were then incubated for 30 minutes at roomtemperature in the dark with periodic vortexing. A second 2.5 μL aliquotof freshly prepared EDC solution was then added followed by anadditional 30 minute incubation in the dark. Following the second EDCincubation, 1.0 mL of 0.02% Tween-20 (BioShop) was added to each beadmix and vortexed. The microspheres were centrifuged, the supernatant wasremoved, and the beads were resuspended in 1.0 mL of 0.1% sodium dodecylsulfate (Sigma). The beads were centrifuged again and the supernatantremoved. The coupled beads were resuspended in 100 μL of 0.1 M MES pH4.5. Bead concentrations were then determined by diluting eachpreparation 100-fold in ddH₂O and enumerating using a NeubauerBrightLine Hemacytometer. Coupled beads were stored as individualpopulations at 8° C. protected from light.

The relative oligonucleotide probe density on each bead population wasassessed by Terminal Deoxynucleotidyl Transferase (TdT) end-labellingwith biotin-ddUTPs. TdT was used to label the 3′-ends of single-strandedDNA with a labeled ddNTP. Briefly, 180 μL of the pool of 100 beadpopulations (equivalent to about 4000 of each bead type) to be used forhybridizations was pipetted into an Eppendorf tube and centrifuged. Thesupernatant was removed, and the beads were washed in 1× TdT buffer. Thebeads were then incubated with a labelling reaction mixture, whichconsisted of 5× TdT buffer, 25 mM CoCl₂, and 1000 pmol ofbiotin-16-ddUTP (all reagents were purchased from Roche). The totalreaction volume was brought up to 85.5 μL with sterile, distilled H₂O,and the samples were incubated in the dark for 1 hour at 37° C. A secondaliquot of enzyme was added, followed by a second 1 hour incubation.Samples were run in duplicate, as was the negative control, whichcontained all components except the TdT. In order to removeunincorporated biotin-ddUTP, the beads were washed 3 times with 200 μLof hybridization buffer, and the beads were resuspended in 50 μL ofhybridization buffer following the final wash. The biotin label wasdetected spectrophotometrically using SA-PE (streptavidin-phycoerythrinconjugate). The streptavidin binds to biotin and the phycoerythrin isspectrally distinct from the probe beads. The 10 mg/mL stock of SA-PEwas diluted 100-fold in hybridization buffer, and 15 μL of the dilutedSA-PE was added directly to each reaction and incubated for 15 minutesat 37° Celsius. The reactions were analyzed on the Luminex¹⁰⁰ LabMAP.Acquisition parameters were set to measure 100 events per bead using asample volume of 50 μL.

The results obtained are shown in FIG. 2. As can be seen the MeanFluorescent Intensity (MFI) of the beads varies from 840.3 to 3834.9, arange of 4.56-fold. Assuming that the labelling reactions are completefor all of the oligonucleotides, this illustrates the signal intensitythat would be obtained for each type of bead at this concentration ifthe target (i.e., labelled complement) was bound to the probe sequenceto the full extent possible.

The cross-hybridization of targets to probes was evaluated as follows.100 oligonucleotide probes linked to 100 different bead populations, asdescribed above, were combined to generate a master bead mix, enablingmultiplexed reactions to be carried out. The pool ofmicrosphere-immobilized probes was then hybridized individually witheach biotinylated target. Thus, each target was examined individuallyfor its specific hybridization with its complementary bead-immobilizedsequence, as well as for its non-specific hybridization with the other99 bead-immobilized universal sequences present in the reaction. Foreach hybridization reaction, 25 μL bead mix (containing about 2500 ofeach bead population in hybridization buffer) was added to each well ofa 96-well Thermowell PCR plate and equilibrated at 37° C. Each targetwas diluted to a final concentration of 0.002 fmol/μL in hybridizationbuffer, and 25 μL (50 fmol) was added to each well, giving a finalreaction volume of 50 μL. Hybridization buffer consisted of 0.2 M NaCl,0.1 M Tris, 0.08% Triton X-100, pH 8.0 and hybridizations were performedat 37° C. for 30 minutes. Each target was analyzed in triplicate and sixbackground samples (i.e. no target) were included in each plate. A SA-PEconjugate was used as a reporter, as described above. The 10 mg/mL stockof SA-PE was diluted 100-fold in hybridization buffer, and 15 μL of thediluted SA-PE was added directly to each reaction, without removal ofunbound target, and incubated for 15 minutes at 37° C. Finally, anadditional 35 μL of hybridization buffer was added to each well,resulting in a final volume of 100 μL per well prior to analysis on theLuminex¹⁰⁰ LabMAP. Acquisition parameters were set to measure 100 eventsper bead using a sample volume of 80 μL.

The percent hybridization was calculated for any event in which the NETMFI was at least 3 times the zero target background. In other words, acalculation was made for any sample where(MFI_(sample)−MFI_(zero target background))/MFI_(zero target background)≧3.

The net median fluorescent intensity(MFI_(sample)−MFI_(zero target background)) generated for all of the10,000 possible target/probe combinations was calculated. As there are100 probes and 100 targets, there are 100×100=10,0000 possible differentinteractions possible of which 100 are the result of perfecthybridizations. The remaining 9900 result from hybridization of a targetwith a mismatched probe. A cross-hybridization event is then defined asa non-specific event whose net median fluorescent intensity exceeds 3times the zero target background. In other words, a cross-talkcalculation is only be made for any sample where(MFI_(sample)−MFI_(zero target background))/MFI_(zero target background)≧3.Cross-hybridization events were quantified by expressing the value ofthe cross-hybridization signal as a percentage of the perfect matchhybridization signal with the same-probe.

The results obtained are illustrated in FIG. 3. The ability of eachtarget to be specifically recognized by its matching probe is shown. Ofthe possible 9900 non-specific hybridization events that could haveoccurred when the 100 targets were each exposed to the pool of 100probes, 6 events were observed. Of these 6 events, the highestnon-specific event generated a signal equivalent to 5.3% of the signalobserved for the perfectly matched pair (i.e. specific hybridizationevent).

Each of the 100 targets was thus examined individually for specifichybridization with its complement sequence as incorporated onto amicrosphere, as well as for non-specific hybridization with thecomplements of the other 99 target sequences. Representativehybridization results for target (complement of probe 90, Table I) areshown in FIG. 4. Probe 90 was found to hybridize only to itsperfectly-matched target. No cross-hybridization with any of the other99 targets was observed.

The foregoing results demonstrate the possibility of incorporating the1168 sequences of Table I, or any subset thereof, into a multiplexedsystem with the expectation that most if not all sequences can bedistinguished from the others by hybridization. That is, it is possibleto distinguish each target from the other targets by hybridization ofthe target with its precise complement and minimal hybridization withcomplements of the other targets.

Example 2 Tag Sequences Used in Sorting Polynucleotides

The family of non cross hybridizing sequence tags or a subset thereofcan be attached to oligonucleotide probe sequences during synthesis andused to generate amplified probe sequences. In order to test thefeasibility of PCR amplification with non cross hybridizing sequencetags and subsequently addressing each respective sequence to itsappropriate location on two-dimensional or bead arrays, the followingexperiment was devised. A 24mer tag sequence can be connected in a 5′-3′specific manner to a p53 exon specific sequence (20mer reverse primer).The connecting p53 sequence represents the inverse complement of thenucleotide gene sequence. To facilitate the subsequent generation ofsingle stranded DNA post-amplification the tag-Reverse primer can besynthesized with a phosphate modification (PO₄) on the 5′-end. A secondPCR primer can also be generated for each desired exon, represented bythe Forward (5′-3′) amplification primer. In this instance the Forwardprimer can be labeled with a 5′-biotin modification to allow detectionwith Cy3-avidin or equivalent.

A practical example of the aforementioned description is as follows: Forexon 1 of the human p53 tumor suppressor gene sequence the followingtag-Reverse primer (SEQ ID NO:1171) can be generated:

                          222087                     2220635′-PO4-ATGTTAAAGTAAGTGTTGAAATGT-TCCAGGGAAGCGTGTCACCGTCGT-3′      TagSequence # 3                   Exon 1 ReverseThe numbering above the Exon-1 reverse primer represents the genomicnucleotide positions of the indicated bases.The corresponding Exon-1 Forward primer sequence (SEQ ID NO:1172) is asfollows:

          221873                     2218965′-Biotin-TCATGGCGACTGTCCAGCTTTGTG-3′In combination these primers will amplify a product of 214 bp plus a 24bp tag extension yielding a total size of 238 bp.Once amplified, the PCR product can be purified using a QIAquick PCRpurification kit and the resulting DNA can be quantified. To generatesingle stranded DNA, the DNA is subjected to λ-exonuclease digestionthereby resulting in the exposure of a single stranded sequence(anti-tag) complementary to the tag-sequence covalently attached to thesolid phase array. The resulting product is heated to 95° C. for 5minutes and then directly applied to the array at a concentration of10-50 nM. Following hybridization and concurrent sorting, the tag-Exon 1sequences are visualized using Cy3-streptavidin. In addition to directvisualization of the biotinylated product, the product itself can nowact as a substrate for further analysis of the amplified region, such asSNP detection and haplotype determination.

DEFINITIONS

Non-cross-hybridization: Describes the absence of hybridization betweentwo sequences that are not perfect complements of each other.

Cross-hybridization: The hydrogen bonding of a single-stranded DNAsequence that is partially but not entirely complementary to asingle-stranded substrate.

Homology or Similarity: How closely related two or more separate strandsof DNA are to each other, based on their base sequences.

Analogue: The symbols A, G, T/U, C take on their usual meaning in theart here. In the case of T and U, a person skilled in the art wouldunderstand that these are equivalent to each other with respect to theinter-strand hydrogen-bond (Watson-Crick) binding properties at work inthe context of this invention. The two bases are thus interchangeableand hence the designation of T/U. A chemical, which resembles anucleotide base is an analogue thereof. A base that does not normallyappear in DNA but can substitute for the ones, which do, despite minordifferences in structure. Analogues particularly useful in thisinvention are of the naturally occurring bases can be inserted in theirrespective places where desired. Such an analogue is any non-naturalbase, such as peptide nucleic acids and the like that undergoes normalWatson-Crick pairing in the same way as the naturally occurringnucleotide base to which it corresponds.

Complement: The opposite or “mirror” image of a DNA sequence. Acomplementary DNA sequence has an “A” for every “T” and a “C” for every“G”. Two complementary strands of single stranded DNA, for example a tagsequence and its complement, will join to form a double-strandedmolecule.

Complementary DNA (cDNA): DNA that is synthesized from a messenger RNAtemplate; the single-stranded form is often used as a probe in physicalmapping.

Oligonucleotidex Refers to a short nucleotide polymer whereby thenucleotides may be natural nucleotide bases or analogues thereof.

Tag: Refers to an oligonucleotide that can be used for specificallysorting analytes with at least one other oligonucleotide that when usedtogether do not cross hybridize.

TABLE I No. in Sequence SEQ ID NO: Ex 1 A A A T T G T G A A A G A T T GT T T G T G T A 1  1 G T T A G A G T T A A T T G T A T T T G A T G A 2 —A T G T T A A A G T A A G T G T T G A A A T G T 3 — T G A T G T T A G AA G T A T A T T G T G A A T 4 — T T T G T G T A G A A T A T G T G T T GT T A A 5 — A T A A G T G T A A G T G A A A T A A G A A G A 6 — A A G AG T A T T T G T T G T G A G T T A A A T 7 — G T G T T T A T G T T A T AT G T G A A G T T T 8 — A A A G A G A A T A G A A T A T G T G T A A G T9 — T A T G A A A G A G T G A G A T A A T G T T T A 10 — A T G A G A A AT A T G T T A G A A T G T G A T 11 — T T A G T T G T T G A T G T T T A GT A G T T T 12 — G T A A A G A G T A T A A G T T T G A T G A T A 13 — AA A G T A A G A A T G A T G T A A T A A G T G 14 — G T A G A A A T A G TT T A T T G A T G A T T G 15 — T G T A A G T G A A A T A G T G A G T T AT T T 16  2 A A A T A G A T G A T A T A A G T G A G A A T G 17 — A T A AG T T A T A A G T G T T A T G T G A G T 18 — T A T A G A T A A A G A G AT G A T T T G T T G 19 — A G A G T T G A G A A T G T A T A G T A T T A T20 — A A G T A G T T T G T A A G A A T G A T T G T A 21 — T T A T G A AA T T G A G T G A A G A T T G A T 22 — G T A T A T G T A A A T T G T T AT G T T G A G 23 — G A A T T G T A T A A A G T A T T A G A T G T G 24  4T A G A T G A G A T T A A G T G T T A T T T G A 25 — G T T A A G T T T GT T T A T G T A T A G A A G 26 — G A G T A T T A G T A A A G T G A T A TG A T A 27 — G T G A A T G A T T T A G T A A A T G A T T G A 28 — G A TT G A A G T T A T A G A A A T G A T T A G 29 — A G T G A T A A A T G T TA G T T G A A T T G T 30 — T A T A T A G T A A A T G T T T G T G T G T TG 31 — T T A A G T G T T A G T T A T T T G T T G T A G 32 — G T A G T AA T A T G A A G T G A G A A T A T A 33 — T A G T G T A T A G A A T G T AG A T T T A G T 34 — T T G T A G A T T A G A T G T G T T T G T A A A 35— T A G T A T A G A G T A G A G A T G A T A T T T 36 — A T T G T G A A AG A A A G A G A A G A A A T T 37  7 T G T G A G A A T T A A G A T T A AG A A T G T 38 — A T A T T A G T T A A G A A A G A A G A G T T G 39 — TT G T A G T T G A G A A A T A T G T A G T T T 40 — T A G A G T T G T T AA A G A G T G T A A A T A 41 — G T T A T G A T G T G T A T A A G T A A TA T G 42 — T T T G T T A G A A T G A G A A G A T T T A T G 43 10 A G T AT A G T T T A A A G A A G T A G T A G A 44 — G T G A G A T A T A G A T TT A G A A A G T A A 45 — T T G T T T A T A G T G A A G T G A A T A G T A46 — A A G T A A G T A G T A A T A G T G T G T T A A 47 — A T T T G T GA G T T A T G A A A G A T A A G A 48 — G A A A G T A G A G A A T A A A GA T A A G A A 49 — A T T T A A G A T T G T T A A G A G T A G A A G 50 —G T T T A A A G A T T G T A A G A A T G T G T A 51 — T T T G T G A A G AT G A A G T A T T T G T A T 52 — T G T G T T T A G A A T T T A G T A T GT G T A 53 — G A T A A T G A T T A T A G A A A G T G T T T G 54 — G T TA T T T G T A A G T T A A G A T A G T A G 55 — A G T T T A T T G A A A GA G T T T G A A T A G 56 — T T G T G T T T A T T G T G T A G T T T A A AG 57 — A T T G T G A G A A G A T A T G A A A G T T A T 58 — T G A G A AT G T A A A G A A T G T T T A T T G 59 13 A T G T G A A A G T T A T G AT G T T A A T T G 60 — G T T T A G T A T T A G T T G T T A A G A T T G61 — G A T T G A T A T T T G A A T G T T T G T T T G 62 14 T G A A T T GA A A G T G T A A T G T T G T A T 63 — G A T T G T A T T G T T G A G A AT A G A A T A 64 — A A A T T T G A G A T T T G T G A T A G A G T A 65 —G T A A T T A G A T T T G T T T G T T G T T G T 66 — G T T T G T A T T GT T A G T G A A T A T A G T 67 — A T G T A G T A G T A G A T G T T T A TG A A T 68 — T G T T T A A A G A T G A T T G A A G A A A T G 69 — T G TG A T A A T G A T G T T A T T T G T G T A 70 — A T A G T T G T G A G A AT T T G T A A T T A G 71 — A T A G A T G T A A G A G A A A T T G T G A AA 72 — A G A T T A A G A G A A G T T A A T A G A G T A 73 — G A A G T AA A T T G T G A A T G A A A G A A A 74 — A A T G T A A G A A A G A A G AT T G T T G T A 75 — T T T G A T T T A T G T G T T A T G T T G A G T 76— G T A T T G A G A A A T T T G A A G A A T G A A 77 — G A A T T G T A TG A A A T G A A T T G T A A G 78 — T A T T G T A G A A G T A A A G T T AG A A G T 79 — T T T A T G T A A T G A T A A G T G T A G T T G 80 — A TA T A G T T G A A A T T G T G A T A G T G T 81 — A T A A G A A A T T A GA G A G T T G T A A A G 82 — G A A T T G T G A A A T G T G A T T G A T AT A 83 — A A A T A A G T A G T T T A A T G A G A G A A G 84 — G A T T AA A G A A G T A A G T G A A T G T T T 85 — T A T G T G T G T T G T T T AG T G T T A T T A 86 — G A G T T A T A T G T A G T T A G A G T T A T A87 — G A A A G A A A G A A G T G T T A A G T T A A A 88 — T A G T A T TA G T A A G T A T G T G A T T G T 89 — T T G T G T G A T T G A A T A T TG T G A A A T 90 — A T G T G A A A G A G T T A A G T G A T T A A A 91 —G A T T G A A T G A T T G A G A T A T G T A A A 92 — A A G A T G A T A GT T A A G T G T A A G T T A 93 17 T A G T T G T T A T T G A G A A T T TA G A A G 94 — T T T A T A G T G A A T T A T G A G T G A A A G 95 — G AT A G A T T T A G A A T G A A T T A A G T G 96 18 T T T G A A G A A G AG A T T T G A A A T T G A 97 — A T G A A T A A G A G T T G A T A A A T GT G A 98 — T G T T T A T G T A G T G T A G A T T G A A T T 99 — T T T AA G T G A G T T A T A G A A G T A G T A 100 19 G A T T T A T G T G T T TG A A G T T A A G A T 101 — T A G T T A G A G A A A G T G A T A A A G TT A 102 — G T A A T G A T A A T G A A G T G T A T A T A G 103 — A A T GA A G T G T T A G T A T A G A T A G T A 104 — T A A A T T G A G T T T GT T T G A T T G T A G 105 — T A A T G A A G A A T A A G T A T G A G T GT T 106 — A A A T G T A A T A G T G T T G T T A G T T A G 107 — A G A GT T A G T G A A A T G T T G T T A A A T 108 — G A A A T A G A A A T G TA T T G T T T G T G A 109 — A G T T A T A A G T T T G T G A G A A T T AA G 110 — G A G T T T A T A G T T A G A A T A T G T T G T 111 — A G A GT T A T T A G A A G A A G A T T T A A G 112 — G A G T T A A T G A A A TA A G T A T T T G T G 113 — A T G A T G A A T A G T T G A A G T A T A TA G 114 — A T A G A T A T G A G A T G A A A G T T A G T A 115 — T A T GT A A A G A A A G T G A A A G A A G A A 116 — T G A A T G T A G A A A TG A A T G T T G A A A 117 — A A T T G A A T A G T G T G T G A G T T T AA T 118 — A G A T A T T G T T T G A T T A A T G A A G A G 119 — A A A GT T G T A A A G T T G A A G A T A A A G 120 — G T T A A G A G A T T A TG A G A T G T A T T A 121 — A G A A G A T A T A A G A A G A T T G A A TT G 122 — G T A G A A A T T T G A A T T G A T G T G A A A 123 — A A G AG T A G A T T G A T A A G T A T A T G A 124 — T G A T A T A G T A G T GA A G A A A T A A G T 125 22 A G A T A A T G A T G A G A A A T G A A G AT A 126 — A T G T G A A A G T A T T T G T G A T A T A G T 127 — A A T AA G A G A A T T G A T A T G A A G A T G 128 23 T A A G T G T A T T T A GT A G A A T G A A G T 129 — T A T G T T A G A T T T G T T G A G A T T GA T 130 — A G T T T G T A T G A A G A G A T A G T A T T T 131 — G A G AA A T G T T A T G T A T T T A G T A G T 132 — T A T G T G A G A A T G TG T T T G A T T T A A 133 — G T A T G T T T G T T T A T A G A A T G T AT G 134 — G A G T A T A T A G A A G A A A G A A A T T T G 135 — A T G AG T G A A G T A A A T G T A G T T A T T 136 — T T A A G A A G T G A G TT A T T G T G A T A T 137 — A T G A A A T G A G A A T A T T G T T G T TT G 138 — G A T T A A T G A T T A T G T G A A T T G A T G 139 — G A A AT G T T A A A G A T A T G A A A G T A G 140 — T A T T G T T G A T T T GA T A T T A G T G T G 141 — T T T A T G T T T G T G T A T G T A A G T AG T 142 — A A T T G A A A G A A T T G T G T G A A T T G A 143 — T G A GT T T G A A T T T G T T T G A G T A A T 144 — G A T G T A T A A T G A TG T G T G T A A A T T 145 — A T G T G A G A G A A G A A T T T G T T T AT T 146 — G T G A T A A A G T A T T G T T G A T A G A A A 147 — G A A GT A G A A T A G A A A G T T A A T A G A 148 — T T G T G T A G T T A A GA G T T G T T T A A T 149 24 T A G T A G T A A G T T G T T A G A A T A GT T 150 — A A T T T G A A G T A T A A T G A A T G T G T G 151 — T A G AA A T T G T A G T A T T T G A G A G A A 152 — T G T A T A T G T T A A TG A G A T G T T G T A 153 25 T A T T T G A T A A G A G A A T G A A G A AG T 154 26 T T G A A T A G T G T A A T G A A T A T G A T G 155 — G T A GT T T G T G A A T A G A A T T A G T T T 156 — A A A G A T G A T T G T AA T T T G T G T G A A 157 — G A A G A T T G T T G A G T T A A T A G A TA A 158 — A G A T T A T G T A G T G A T G T A A A T G T T 159 — G A A TT T A G A T G T A G A T A T G A A T G T 160 — G A T A G A A G T G T A TT A A G T A A G T T A 161 — T A T G A A T T A T G A G A A G A A T A G AG T 162 — T T T G T T A T G A A G T G A T T T G T T T G T 163 — G T A AA G A T T G T G T T A T A T G A A A T G 164 — T T G T G A T A G T A G TT A G A T A T T T G T 165 28 G A A T T A A G A T A A A G A A G A G A A GT A 166 — G A T T G T A G A A T G A A T T T G T A G T A T 167 — A A A TA A G A G A G A G A A T G A T T T A G T 168 — A A T T A T G T G A A T AG A T T G T T G A A G 169 — T T A A G A T T T A T G T G A T A G T A G AG T 170 — T T A A A G A T A G T G T T T G T T G T G T T A 171 — T A T TG A T T T A T G A A G A G T A T A G T G 172 — A A A T T T G A T G A G TA G T T T A A G A G A 173 — A T A A A G T T G T T T G A T G T T T G A AT G 174 — G A T T G T G A T G A A T A A T G T T A T T G A 175 — G A T GA A G A A A T A T G A T A T G A A T A G 176 — T T A A A G T T A T T G AA G T G A A G T T G A 177 — T T G T A A G A A A T A G A G A T T T G T GT T 178 — G A G A T T G A G T T T A A G T A T T A G A T T 179 — A G T GA T A A T A G A A T G A T A A A T G T G 180 — G A T A A T A G T G A A TT T G A G T T G T A T 181 — A G A T A T T T G T A G T A G A A A G T A TG T 182 — G T T A T G A A T G T T G A A T T T G A A T G T 183 — A T G AA A G A T T T A G T T G T G A G A T A T 184 30 A A A T A G A G A A G T TA T G A T G T G A T A 185 — T T A G T G A G A A A T G T T T A A T G T GA T 186 — T G A A G A A T A T G T G A A A T T A G T T T G 187 — G T T TG A T A G T T T A A T G A G T A T T G A 188 — G T T G T A A G T A A T GA T A A A G T A T G A 189 — T A A G A G T A G T A A T T G T T G T T T AG A 190 — T T T G A G A G A G T A T G T A T G A T T A T T 191 — A T T GA T T G T G A A T T A G A T A G A A G A 192 — G A T T A G T A T T T A GT A G T A A T A G A G 193 31 T A T G T A T T A G A G A T A T T G A A A GT G 194 — T A T G T G A A A G T A A T G A T A A A T G A G 195 — G T A AT T A G T A A T G A T T T G A A T G A G 196 — G T T T A T T G T A A A GA T G T A A G T G A A 197 — T A G T A G A A T T G T T G T T A A A G A AT G 198 32 T A T T G T T A G T T A T G T A G T G T G T A A 199 — G A G TG A A A G T T A T A T G A A A G T A T A 200 — A T A T A G A A G T T G AT G A G T T T A T G A 201 — T T T A G A A G T A A G A A T A A G T G A GT A 202 — T G T G T A T A A G A T A T T T G T A A G A A G 203 — T A G AA G A G T T G T A T T G T T A T A A G T 204 — G T G T T A T T A G T T TA A G T T A G A G T A 205 — A A T A T A G T G A T G T G A A A T T G A AT G 206 — T T A G A G A A T A G A G T G A T T A T A G T T 207 — G A A GT G A G T T A A T G A T T T G T A A A T 208 — A A T G T A A A G T A A AG A A A G T G A T G A 209 33 G T T A G T T A T G A T G A A T A T T G T GT A 210 34 A A A T G A G T T A G A G T A G A A T T A T G T 211 — G A T AT A G A A G A T T A G T T A G T G A T A 212 — A T A G T T T G T T G A GA T T T A T G A G T A 213 — T A G A A T A G T T A G T A G T A A G A G TA T 214 — G A A T T T G T A T T G T G A A G T T T A G T A 215 — G T A GT A A G A A G A G A A T T A G A T T A A 216 — A A T G T G T T A T G T AT G T A A A T A G T G 217 — G A A T T A G T T A G A G T A A A T T G T TT G 218 — G A A A T T G A A G A T A G T A A G A A A T G A 219 — G T G TA T T A T G T G A T T T A T G A T A G A 220 — T A T T A T G A G A A A GT T G A A T A G T A G 221 35 T A T G T A T T G T A T T G A G T A G A T GA A 222 — G T G A T T G A A T A G T A G A T T G T T T A A 223 36 A G T AA G T T G T T T G A T T G A A A T T T G 224 — G A A G T T T G A T T T AA G T T T A A G A A G 225 — G A G A A G A T A A A T G A T A T T G T T AT G 226 — A T G A T G A G T T G T T A A T A G T T A G T T 227 — T A T GA T A T T T G A A G A G T G T T A A G A 228 — G A G A T G A T T A A A GT G A T T T A T G A A 229 — A T A G T T A A G A G T G A T G A G A A T AA A 230 — T T T A T T G T T A G A T A A A G A G T T G A G 231 — A G A AT A T T G A T A G T T G A A G T T G A A 232 — T A G T G T A A A G T G TA G A T T G T A A A T 233 — A G T A G T G A T A T G A T T T G A A T A TT G 234 — T G T A T T G A A T T A G A A T A G T G A G A A 235 — T G A TA T G A G A T A G A A G T T T A A T G T 236 — G A A G A A G T A A G T AT A A A G T A A A T G 237 — T T T A A G T G T G A T A A G A A A G A T AG A 238 — T A T T G T T G A A T G T G T T T A A A G A G A 239 38 G A A TA A T G A T G A G A T G A T T A T T G A 240 — T A G A G A A A G A G A GA A T T G T A T T A A 241 39 A T G T A T A A T G A G A T A T G T T T G TG A 242 — A A T A G A T A A G A T T G A T T G T G T T T G 243 40 T T T GA T G A T A A T A G A A G A G A A T G A 244 — A G A T G A A T A A G T TG T G A A T G T T T A 245 — A G A T G A A A G A A A G T G T A G A A T AT T 246 — T G T T A A A T G T A T G T A G T A A T T G A G 247 41 T A G TA G T G T G A A G T T A T T T G T T A T 248 — A G T G A A T G T T T G TA A A G A G T T T A A 249 — G A T A A A T G A G A A T T G A G T A A T TG T 250 — T G A T G A G A A A T T G T T T A A G T G T T T 251 — A A A TA A G T A G T G T G A G T A A T A G T A 252 — T A T G A A A T A T G T GA T A G T A A G A G A 253 — A T T G T A A G A G T G A T T A T A G A T GA T 254 — A G A G T A A G A A T G A A A G A G A T A A T A 255 — T A A GT A A G T A G A T G T T A A A G A G A T 256 — A A A T A G A A A G A A TT G T A G A G T A G T 257 — A T A G A T T T A A G T G A A G A G A G T TA T 258 42 G A A T G T T T G T A A A T G T A T A G A T A G 259 43 A A AT A G A A T G A G T A G T G A A A T A T G 260 — T T G A A T T A T G T AG A G A A A G T A A A G 261 — T A G T A A A T T G A G A G T A G T T G AA T T 262 — T G T A A A G T G T T T A T A G T G T G T A A T 263 — A T AT G A T T T G A G A T G A G A A T G T A A 264 — A A T A T T G A T A T GT G T T G T G A A G T A 265 — A G T G A G A T T A T G A G T A T T G A TT T A 266 44 T T G T A T T T A G A T A G T G A G A T T A T G 267 — A T AG A A A T G A A A G A T A G A T A G A A G 268 — G A T T G T A T A T G TA A A G T A G T T T A G 269 — T A T G A A T G T T A T T G T G T G T T GA T T 270 45 G A T A T T A G T A G A G T A A G T A T A T T G 271 — T G AG A T G A A T T T G T G T T A T G A T A T 272 — T A T G A A T G A A G TA A A G A G A T G T A A 273 — G A G T G A A T T T G T T G T A A T T T GT T T 274 — A G A A A T T G T A G A G T T A A T T G T G T A 275 — G T GT T A A T G A A A G T T G T G A A T A A T 276 — T G T G A T T T G T T AA G A A G A T T A A T G 277 — A G T A G T A T T G T A A A G T A T A A AG A G 278 — T G A T T G T T G T A T A G T T A T T G T G T A 279 — G A TT G T A G T T T A A T G T T A A G A A T G 280 — A T G A A A T A A G A AA T T G A G T A G A G A 281 — T A T G A T G A T A T T T G T T G T A T GT G T 282 — T T T A G A G T T T G A T T A G T A T G T T T G 283 — A A TA A G A G A T T G T G A T G A G A A A T A 284 — A A T G A A T A G A A TA G A G A A T G T A G A 285 — G T A G T A G T A A T T T G A A T G T T TG A A 286 47 A G T G A G T A A T T G A T T G A T T G T T A A 287 — G A AT A A T G T T T A G T G T G T T T G A A A 288 — A T A T G A A A G T A GA G A A A G T G T T A T 289 — T G A G T T A T T G T A T T T A G T T T GA A G 290 — T A G T T G A G T T T A A A G T T G A A A G A A 291 — T A AA G A G T G A T G T A A A T A G A A G T T 292 — T G T A G T G T T T A GA G T A A G T T A T T A 293 — A G A G A T T A A T G T G T T G A A A G AT T A 294 — G T A A T A A G T T G T G A A A G A A G A T T A 295 — G A GA T G T T A T A G A T A A T G A A A G A A 296 — T T T A G T T G A T T GT T G A A T A G A G T A 297 — A T T A T T G A A A G T A G A T G T T A GA T G 298 — T T T A T G T G T G A T T G A G T G T T T A A T 299 — T A TT T A G T T A G A T A G A T A G A G A G T 300 — A T G T G T T T A T G TG A A A G A T T T G T A 301 — A T A G T A A T T A G A A G A G A A G A AT G T 302 — T A T G A G T G A T T A G A A T T G T A T T T G 303 — T T AA T G T A T T G T T T A A A G A G T G T G 304 — A T A G A G A A T T A AG A A T T G T T T G A G 305 — G T T A T A A G T A G A A A T G T A T A GA A G 306 — A G T A A T T A G T T T G A A A T G T G T A G T 307 — G A AA G A T T A T G A T T G T A A A G T G A T 308 — G T A A G A T T A G A AG T T A A T G A A G A A 309 48 G A G A A T G T T G A A T A A G A A G T AA T T 310 — T T A A G A G T G T T T G A A T A G T G T T T A 311 — A T AA A G A A A G A G T A T G A G A T T A T G 312 — A G T T A T T G A T T GA A G A T G A G A A A T 313 — G T T T G T G T T T G T A T A A G T T G TT A A 314 50 T T G T A T G T G A G T T T A G A T T A A T G A 315 — T A GT T A A A G T A T A G T T G T T T G A G T 316 — A A A T T T G T G T T GA G A T T T G T A T A G 317 — T A T T A G T G T T A T G A T A A A G A GA A G 318 — T A T A A G A A G T A A T T T G A G A A G A G T 319 — T A AG T T G A G A T G T T T G T T T G A T A A 320 — G T G T A G A T T T A TG A A T T G A G T A A T 321 — T A T A G A G A A G T G T T T A G T T G TA T A 322 — A T A A A G A A G A A T A G T T G T T G T G T A 323 — A G AT T G A A A T A G A T T A G A A A G T T G 324 — G T T G T T A T A A G AA A T A G T T T G T T G 325 — A G A A A T A G A G T A A G A G T G T T TA A A 326 — A G A G A T A G T A G T A A A T A G T T A T T G 327 — A A AT G A T T G T G T A A G T T A T G T A T G 328 — A A G A A G T A A G A GA G A A A T T T G A A T 329 — G T G T G T A T T T A G T T G A T A A T TG A T 330 — A T T G T T G T T G T T G A G A A A T G T A T T 331 — A G AT A A G T T A A A G T A A A G A G A A T G 332 — T A G T T G A A G T T AG T T T A A G T G T T A 333 — A G T A A G A A T G T A A T A T G A T G AT A G 334 — A T G A G A T T G A A A G A T T T A T G A A T G 335 — T G AT T G A A T T A G A G A G A A T G T A T A 336 — A G T T A G T A A G A GA A T A T A G T G A A T 337 — A T T A A G A T T G T A T A G T T A G T GA T G 338 — G A G A T A A A G A A T T G A A A T A G A A G A 339 — A G AG T A A A T G T T A A G A A A G A A G T T 340 — A A A G T T T G T T A TG T G T G A A G A A T T 341 — A T T G T G T T T A A G A A A T A T G A TG A G 342 — T A T T G A A A T G A G A T G T A T G T A G T T 343 — A T TT G T G T G A T G T T T G A A A T A T G A 344 — T A A G A T A A T A G TG A G A G A A A T T G A 345 — A T T T A T G A T T A G T G T A A G T G TT G T 346 — G A T T A A G A A T A A A G T G T G A A G A A T 347 — G T AA T T G A T G A A G A G T T A G T T T A T 348 — T G T G T T A T G T T AT A A G A A G T G A T A 349 — A G A G A A A T T G A A T T T A G A A A TG T G 350 — T T A T T G A A T G T G A G A A A G T A T T T G 351 — T G TT A A T G A G A A G A T A A T G A T A G T 352 — G A A A G T A T T T G TT G A T T A T T G T T G 353 — T A G T T T A T G T A G T T A A T T G T TG A G 354 — G T T G A A A G A T A G T T T G A T A T G T A T 355 — T T AG A A G A T A G A T T A T T G A G A A A G 356 — A A T A A T G T T G T GA A A T A G A T G T G A 357 56 A G T A A G A A A G T T T A G T T T A G TT A G 358 — T A G T T T A A T G A G A T G T T T G A T A T G 359 — T T AA A G A T G T T A A A G A A T G A G T G A 360 — A A A G T G T G T A T AT G T T A G A A A G T A 361 — A T T A A G T T A T G T G T T T A T G T GT T G 362 — T T T G A A G A A G T G T T T G T A T T A T G T 363 — T G TT A A G A A G T T T A G T T A A A G T T G 364 — T T T A A G T A T A A GA T T G T G T G A G A T 365 — A G A T A T T T G A T A G A T A G A A G AA A G 366 — A T T T A G A G T T G T A A G A A G A T A T T G 367 — G A GA A A T T G T A A T T G T T A G A G T A T 368 — G A A G T A T A T G T TA A G A T G T A A T A G 369 — A A T A T T G A A G A T G T A G T G A G TT A T 370 — G A G T T T A G A A A T G A T A A A G A A T T G 371 — T A AG A A A T G A G T T A T A T G T T G A G A 372 60 T T G A T A T A A G A AG T T G T G A T A A G T 373 — A A G T G T T T A A T G T A A G A G A A TG A A 374 61 G T T G T G A G A A T T A G A A A T A G T A T A 375 — T T TA G T T T G A T G T G T T T A T G A G A T 376 — G T A A T T G A A A G TA T G A G T A G T A A T 377 — T A G T T G A A T A A G A T T G A G A G AA A T 378 — T T A A G T G A A G T G T T G T T T A T T G A A 379 — A T TG A T T T G T T G A A A T A A G T G T T G 380 — T G A A T T G T T G A TA A G T T A T G A A G A 381 — G T T T G T T A T T G A G T A A G T T G AA T T 382 — T G A T T T A G T A T G T A T T A G A G T T G A 383 — T A AA T A G A G A T G A G A A T A A G A A A G 384 — A G A A T G T T A T A TG T A G A G A A A T T G 385 — A T T T A T G T A G T T G A G A G T G A TA A A 386 — G T A A A G A T A G T T T G A G T A A T T T G A 387 — G A AA T A G T A T A A T G T T A A G T G A G A 388 — A T T G T A T A T T G TG T T G A A G A A A G T 389 — G A G T T A A G T G T A A A T G A A A T GT A A 390 — A T A G A T T G T G T G A A A G A A A G A A T T 391 — T T AA T A G A A G T T T G T A G T A T G A T G 392 — T T G T A T G T G A G AA T A A A G T T T A G T 393 — G T G A T T A G A T A T G A T G A T A T GA A T 394 — T G A A G A A G A A T T T A G A T T T G T A A G 395 — T G TA T G A T T A T T G A T T A G T G T G T T 396 — T G T G A A A G A G A AT G A T A G A T A T T T 397 — A A T T G A A A T G A G T G T G T T T A AG A A 398 — A T T A T A G A G T T A G T T T A G A A T G A G 399 — A A AG A T A G A A A T T G A G T G T A T G A T 400 — G T A G T T T G T T A AT G T T G T A T A A T G 401 — A G A G A T A T T A G A A T G T A A G A AT A G 402 64 A G A A G T T T G A A A T A T G A T A G A A T G 403 — T A GA A T G T A A A G T T T A G T A T A G A G 404 — A G T A G A T G T A T GT T A A T G T G A A T A 405 — T G A A A G T G A A A T A T G A A A T G TT G T 406 — A T A G T A T A T T G A G T T T G T A T G A A G 407 — G A AG A A A T G T T T G T A G A A T A A G T A 408 — A A T G A G T A T T G AA G A A A T G T A T A G 409 — G T G A T A G A A T T T G T G T T T A A TG A A 410 66 T G T A G T A T G A A G A A T A A T G A A A T G 411 — A T AG A A G T T A A T G A T A A T T G T G T G 412 — G T G A T T G T A A G TA A G T A A A G A T A A 413 — T A T G T A G T T T G T G T T A T T T G AA G A 414 — T G A G T A A G T T T G T A T G T T T A A G T A 415 67 T A AA T G T A T G A G T G T G T A A A G A A A 416 — G T A A G A G T A T T GA A A T T A G T A A G A 417 68 G T T G A G T G T A A A G A T T A T T G AT A A 418 — A G T A T G A G T T A T T A G A T A A A G T G A 419 — A T TT G T T A T A G A G T T G T G T T G T A T 420 — T A A T T A G T A G T GT G T T G A A A T T T G 421 — T G T A T T G A G A T T G T T A T T G T AT T G 422 — G T T A T T A G A A G A G A T A A T T G A G T T 423 — T T GA G T T G T G A T T A A G T A G T A T A T 424 — G A T A G T A T A A T GA T T G A A G T A A T G 425 — G T G A A A G A T A T T T G A G A G A T AA A T 426 — A G T T A T G A T T T G A A G A A A T T G T T G 427 — G T AA G T A T T T G A A T T T G A T G A G T T 428 — T A A T A G T G T T A TA A G T G A A A G A G T 429 — A A A T G A A T T G A T G T G T A T A T GA A G 430 — A G A A A G T G A G T T G T T A A G T A T T T A 431 — T T TA T G T G T G A A T T G T G T A T A T A G 432 — G T A A T A T G A T A GA A A T G T A A A G A G 433 — G A G A A T T G T T T A A A G A T A G T TG T A 434 — G A A T T T G T T A A G A A T G A G T T T G A T 435 — A T AG T G A T G A T T A A A G A G A A T T T G 436 — A T A G A T G T T T A GT T G A G A T T A T T G 437 — A A G A G T G T A A A T A G A A A G T G AT A T 438 — T G T G T A T T G A T T G T T G A G A T A A A T 439 — T A GT A T A G T G A G A A A G A G T T A A A T 440 — A A A G A T A A G A A AG A G A T G A T G T T T 441 — G A A G T T A T T G A A A T A G A G A A GT A T 442 — A T G T A T G T A T A G A A A G A G T A A A T G 443 — G A TG T T T G T A A A G A T T G A A A T T G A 444 — A A T T T A G A G A G TA T T T G T G T T G T A 445 — A A T T T G T T T G A A A G A A A G T A AG T G 446 — A A A G A G T A G T G T T A T T G T T A G A T A 447 — G T AT G T T G T A T A T G T T G T T G A T A T 448 — G T A G A A T T T G T TG A G T A T T T G T A A 449 — A T G A A T T T A G T T A G T G T A A G AA A G 450 — A T G A T A A G A A A T G T T G A T G A A G T A 451 — T T GA T G A T G A A G A T A A T G T A G A T A 452 — A G A T G A T A T G A TA T A G A T T A G A T G 453 — T T G A A A G T T A G A A A G A T A G A TG T T 454 — G T T T A A T G T T A G T T A G A A A G T A A G 455 — G A GA T T T A A G T T T G A A G T G A A A T A 456 — T T T G T T A G T A G TT G T T A T A A G A G A 457 — T A T G A G A A T A G T T T G T T A G T GA A T 458 — T T G A A A G T T T A A A G A A G A G A T A A G 459 — A A GT G A G T T G A A A T G A A A T A T G T T 460 — G T T A G A A A T G A AA T G A G T A G T T A T 461 — T A A G T A T T G T A T T T G T G T G T GT A T 462 — T G T A T T A G T A A A G A A G A G A G A A T A 463 — G A GA A G A G A A A T A A G T T G A A A T A A 464 — G T A A A G T A G A A AT A G A A T T G A G T T 465 — G T G T G T T A T T T G T T T G T A A A GT A T 466 69 T T T G A T G T A T G A A T A T A G T A T G A G 467 — A A GA T T G T G T G A A T A G T T G A A A T T 468 — T A T A A A G T T T G AA G A T G A G T G A T A 469 — A G A T A A A G A G A T T T A A G A T G TA T G 470 71 G A A G A A T T A A G T T G A G A A T T A A G A 471 — T A GA G A A A T T T G A T A A A G A A A G A G 472 — A A A G T T T A T G A AG T T A T T G A G T A G 473 — A A A T A G T G T A A G T A A A G A G A TG A T 474 — T A T G A T G A T T T A G T T A T A A G A G T G 475 — T A GA T A A A T G T T A T G A T G A G T A A G 476 — A G A T T G A T T G T GA T G A T T T G T A T A 477 — T T A A G A A G A A T T G T A T A T G A GA G T 478 73 G T A G A A T G T T T A G A G T T G A A T A T A 479 — G A GA A A T A G T A A G A A G T A A A T A G A 480 — A T T G A A G T T G T TA T G T G A A G A T T T 481 — T A A A T G T T G T G T A G A G T A A T TA G A 482 — A A A T A A G A G T T T G A G A A G T T G T T T 483 — A G TT G T A A T A A G A A G T G A T T T A A G 484 — G T T A G A A T G T A TA T A G A G T T A G A T 485 74 T T G A T A T T G A A A G A G A A A G T TA T G 486 — T T A A A G A G A G A A A T G T T T G A T T A G 487 — T G TG A A T T T G A G T A T T A G T A A G A A 488 — T A A T T T G A A T G TG A A A G T T G T T A G 489 — A T G T G T T T G A A A G A T G A T G A TT T A 490 — A A G T T A T G T T G A T A T T G A G T G A A A 491 — T A GA T A A A G A A G A T A G A G A T T T A G 492 — G A T G A A T G T A G AT A T A T G T A A T G A 493 — G A A G A A T A G T T T A T G T A A A T GA T G 494 — G T A G T A T A T A G T T A A A G A T G A G T T 495 — G T TA T T T G T G T A T G A T T A T G A T T G 496 — A G A G A T T A G A A AT T G A G A G A A T T A 497 — G T A T G A T A G A G T T T A T A G T G AT A A 498 — G T T A G A A A G A A T G A A A T T G A A G T A 499 — A A GA A T G A G A A T A T A G A G A T G A A T 500 — A A A G A G A A T A G TG T T T A A G A A G A T 501 — G A T G T G T T A T T G A T A G A A A T TA G A 502 — T A G A G T T A T A G A G A T A T T G T A T G A 503 — G A GA G T T G A A T A A G T T A A A G A T A T 504 — A G A T A T G A A A T AG A T T G T T A G A G A 505 — G A G T G A A T A G A A A G A T A T G T TA A T 506 — A A A G A G A T A T T G A A G A G A A T A A A G 507 — G T TA T A G A A T A A G T T G T A A A G T G T 508 — T G A T A G T A T G A TA A T G T G T T T A T G 509 — T T T G T T G T T A A G T A T G T G A T TT A G 510 77 T A A A G T G T T G T G T T A A A G A T T A A G 511 — T G TG T T T G A T T G A T T A A T G T T A T G 512 — A T T A A T G A A T G AG T G T T G T A A T G T 513 — T A G A T G T T T G T G A G T T T G A T AT T A 514 — G A A T G A A T A G T A A T A G A T G A T T T G 515 — A A TA G T G T G T T G T T A T A T G A T T A G 516 — T A G A T T A G A A G AT G T T G T G T A T T A 517 — A A T G T G T G T G T T A A A T G A A T TT G T 518 — G A A T T A A G T A T A T G A G T G T A G A A A 519 — T T AT T G T G T G T A A G T A G T G T A A A T 520 — G T A G T A A A G A G AA T T G T T T A G T A T 521 80 A A G T T T G T A A G A A G T A G T T G AA T A 522 — A G T T A T A G T A T A G T A G T A T A G A G A 523 — G A AA G A A A T G T G T A T A G T T T A A T G 524 — T T G T G A G T A A T GA A T G A T G T A T T A 525 — G T A G A G T T G T A A A T A G A G A A TA A A 526 — A T T A A T G T A G A T T G T A A G A G A T A G 527 — T T AG T G T G T T T G T A G A T A G A A T T A 528 — A G A G A G T T T G T GT A T A T G T A T A A A 529 81 T T A A G T T T A G T G A G A T T T G T TA A G 530 — A T G A A G T T T A T T G A A T A G T A G T G A 531 — A T AT T T G T G T T G T A T G T T T G T G A A 532 — A A A G T G T T T A T AG A A G A T T T G A T G 533 — A A G A G A T A T G A T T T G T T A G T TG T A 534 — A A G A A G A A A T G A G T G A T A A T G T A A 535 — T A GT G T T T G A T A T G T T A A G A A G T T 536 — G T A G A A A G T G A TA G A T T A G T A A T A 537 — G A T A A A T G T T A A G T T A G T A T GA T G 538 — A G A T T A G A A G A A T T G T T T A G A A T G 539 — A T AT T T G A G A A G T G T G A A A T G A A T 540 — T G A G T A A A T A G TT T A T G A G T A G T A 541 — T T A G A G A G T A G A T A A A G A T T TG A T 542 — A T T G T T T A A G T T G T T G A T A A G A T G 543 — G T TG T A A A G T T A A A G T G T G A A T T T 544 — A T A G A T T G T G T GT T T G T T A T A G T A 545 — G T A A G T T A T T G A G A A T G A T A AT A G 546 — T A G A T T A G T T G A T A A G T G T G T A A T 547 83 A A AT G T A A A T G A A G A G T G T T T G T T 548 — G A T A G A A G A A A TG T A T A T A G T G A T 549 — T A T A G A G T G T A T G T T A T G A T AA A G 550 — T A T G A A G T G A T A A G A T G A A G A A T T 551 — T G TT G A G A A T A G T A A G A G A A T T T A 552 — T A G A T A A T G T G AA G T A A T A A G T G A 553 84 G T A T T A T G A T G A T A G T A G T A AG T A 554 — A G A T A T G A T T T A G T A T T G A A T G T G 555 — A A TT A A G T T T G T A G A G T G A T T T G A 556 — A A G A A A T A G A T GT A G T A A G A T G T T 557 — T T G A G A A G T T G T T G T A A T A A GA A T 558 — A G T G T G A A A T A G T G A A A G T T T A A A 559 — T T TA T G T A G T A G A T T T A T G T G A A G 560 — A T T A A T G A G A A AT T A G T G T G T T A G 561 — A T G T T A A T A G T G A T A G T A A A GT G A 562 — T A T G T T G A T A A A T G A T T A T G A G T G 563 — T T AT T A G A G T T G T G T G T G A T A T A T 564 — T G T T G T T A T G A TT G A G T T A G A A T A 565 — A A T T T G A G T T A A G A A G A A G T GT A A 566 — A A A G A T A A A G T T A A G T G T T T G T A G 567 88 T G TT G A G A T G A T A T T G T A T A A G T T 568 — T A A A T A G T G A A TG A G T T A T A G A G T 569 — A T A G A T G T T A T G A T A G T T A G TT A G 570 — G T T A A G T G A A G A T A T G T A T T G T T A 571 — T A AG A A A G T A A A G T T T G T A G A T G T 572 — A A G A G A A A G T T TG A T T G A A T A A A G 573 — A T A T T A G A T G T G A G T T A T A T GT G T 574 — A G T T T G A G T T T A G T A T T G T G A A T A 575 — A T GT T A A A T G A G A G A T T G T G T A T A 576 — T A A A T G T T G T G AT T A T T G T G A G A T 577 — T A A G A A T T G A A G T A A G A G T T AT T G 578 — A G A G A T A G A A T T A A G T T T G T T G A T 579 — G A AG A A T G T T A A G A A A T A T G T A A G 580 — T A T T T G T G A T T AA G A A G T T G A G A A 581 — A G T T A G A A T T T G T G T A G T A G AA T T 582 — A A G T T T A T T G T T G A T G T T G T A T T G 583 — G A AT G A G T T T A A G A G T T T A T A G T A 584 — A G T G A A G A T T G TA T G T A G T A T A A A 585 — A G T T G A A A T G A G T A T T A A G T AA T G 586 — A T G T G T T A T T T G A G A T G A G T A A T T 587 — A A AT A G T G T T G T T G A A G T T G T T A T 588 — G T A G A G A A A G A TA T A T G T A G T T T A 589 — G A G A G T A T T T G A T G A A T G A T TA T A 590 — G A G T A T A A G T T T A G T G T A T A T T G A 591 — A T AA T G T G A T T A T T G A T T G A G A G A 592 — T T A G T T G T T A T GT G A G A G T A A T A A 593 — A A A T G A G T A T A T T G A A T T G T GA T G 594 — A A T T A G A A G T A A G T A G A G T T T A A G 595  3 T G TA A G T T T A A A G T A A G A A A T G T G 596  5 G A A A T G A T A A G TT G A T A T A A G A A G 597 — A A T G A G T A G T T T G T A T T T G A GT T T 598 — A G T G A A T G T A A G A T T A T G T A T T T G 599  6 G T AA T T G A A T T G A A A G A T A A G T G T 600  8 T A T G T T T A A G T AG T G A A A T A G A G T 601 — G T A T T G A A A T T G A A T T A G A A GT A G 602 — A A T A T G T A A T G T A G T T G A A A G T G A 603 — T G AA T A T T G A G A A T T A T G A G A G T T 604 — T A G T G T A A A T G AT G A A G A A A G T A T 605 — G T A T G T G T A A A G A A A T T T G A TG T A 606 — A A T T G T T T G A A A G T T T G T T G A G A A 607 — A A TT G T T T G A G T A G T A T T A G T A G T 608 — T A A T T G A G T T T GA A T A A G A G A G T T 609 — T G T T G A T T G T A A G T G T T T A T TG T T 610 — G A A A T T T G T G A G T A T G T A T T T G A A 611 — T A AG A A T G A A T G T G A A G T G A A T A T 612 — T A A T G T G A A G T TT G T G A A A G A T A T 613 — T T G T A T A T G A A A G T A A G A A G AA G T 614 — T A G A G A G A A G A A G A A A T A A G A A T A 615 — A T TT G A A A T G T T A A T G A G A G A G A T 616 — T T G T G T G T A T A TA G T A T T A G A A T G 617 — A T T G T T A G T A T T G A T G T G A A GT T A 618 — T G T T T G T A T T T G A A T G A A A T G A A G 619 — T G TT A G A T T G T G T T A A A T G T A G T T 620 — T A T A G A G T A T T GT A T A G A G A G A A A 621 — A A A T A G T A A G A A T G T A G T T G TT G A 622 — T G A G T G T G A T T T A T G A T T A A G T T A 623 — A G AA T T T G T T G T A G T G T T A T G A T T 624 — G A T T G A A G A A A GA A A T A G T T T G A A 625 — G A T A A T A G A G A A T A G T A G A G TT A A 626 — G A T T G A A A T T T G T A G T T A T A G T G A 627 — G A TT T A A G A A G A T G A A T A A T G T A G 628 — T T T G A G A G A A A GT A G A A T A A G A T A 629 — G A T T A A G A G T A A A T G A G T A T AA G A 630 — T T T G A T A G A A T T G A A A T T T G A G A G 631 — T G AA G A A G A G T G T T A T A A G A T T T A 632 — G T G A A A T G A T T TA G A G T A A T A A G T 633 — A A A T A A G A A T A G A G A G A G A A AG T T 634  9 G T T G T A A A G T A A T A G A G A A A T T A G 635 — A G TG A T T T A G A T T A T G T G A T G A T T 636 — A G A G T A T A G T T TA G A T T T A T G T A G 637 — A T G A T T A G A T A G T G A A A T T G TT A G 638 — A T G A A A T G T A T T A G T T T A G A G T T G 639 — A T AT T G A G T G A G A G T T A T T G T T A A 640 — A G A T G T G T A T T GA A T T A A G A A G T T 641 — T A A T G T G T T G A T A G A A T A G A GA T A 642 — A A A T T A G T T G A A A G T A T G A G A A A G 643 11 T T TA G A G T T G A A G A A A T G T T A A T G 644 — G A T T G T T G A T T AT T G A T G A A T T T G 645 — T G T T G T T G T T G A A T T G A A G A AT T A 646 — A T T A A G T A A G A A T T G A G A G T T T G A 647 12 G T AT G T T G T A A T G T A T T A A G A A A G 648 15 T A G T T G T G A T T TA T G T A A T G A T T G 649 — T G A T A A T G A A A G T T T A T A G A GA G A 650 — G T A A G A T T G T T T G T A T G A T A A G A T 651 — T T GA A T T A A G A G T A A G A T G T T T A G 652 — A A G T G T T T G T T TA G A G T A A A G A T A 653 — A G A G A G A T A A A G T A T A G A A G TT A A 654 — A T T A T G A A T A G T T A G A A A G A G A G T 655 — T T GT T G A T A T T A G A G A A T G T G T T T 656 — T T T A T T G A G A G TT T G T T A T T T G T G 657 — A G T G T T A A G A A G T T G A T T A T TG A T 658 — G A G A A A T G A T T G A A T G T T G A T A A T 659 — G A TA A G T A T T A G T A T G A G T G T A A T 660 — T T T G A T T T A A G AG T G T T G A A T G T A 661 16 A A G T T A G T A A A T A G A G T A G A AA G A 662 — G T A A A G T A T G A A T A T G T G A A A T G T 663 — T A AT A A G T G T G T T G T G A A T G T A A T 664 — A A A G A T T T A G A GT A G A A A G A G A A T 665 — T T A G T T T G A G T T G A A A T A G T AA A G 666 — T A A T A G T A T G A G T A A G A T T G A A A G 667 — G A AG A T T A G A T T G A T G T T A G T T A A 668 — T A A A G A G A G A A GT T A G T A A T A G A A 669 — T A A G T A T G A G A A A T G A T G T G TT A T 670 — G A G T T T G T T T G T T A G T T A T T G A T A 671 — A A GT A A A G A A A T G T T A A G A G T A G T 672 — A T G A G A A T T G T TG T T G A A A T G T A A 673 — T T A G A T T A G A G T A G T A G A A G AA T A 674 — T A G T G A T G A A G A A G T T A G A A A T T A 675 — T A AT G T A G T A A T G T G A T G A T A A G T 676 — T T G A G A A A G A A TA A G T A G T G T A A A 677 — T A A T G A G T G A G A T T A T A G A T TG T T 678 — G T A T A A G A A A T G T G T G T T T G A T T A 679 — G T GA A T G T G T T A A T G A A G A T A T A T 680 — G A A A G T T A T T A GT A G T T A A A G A T G 681 — T A G A A T T G T G T T T G A T A A G T GA T A 682 — T G A T T T A G A T T G A G A G T T A A A T G A 683 — A T TA T T G A G T T T G A A T G T T G A T A G 684 — A T A G T A G T T A T GT T T G A T T T A G T G 685 — A T A G A A G A A G A A T A A A G T T A GA G A 686 — G A T G T T G A A A G T A A T G A A T T T G T A 687 — G A GA T T G A T A G T A G A A A T G A T A A A 688 — T G A G A G A A T A A AG T A T G A A T T T G A 689 — T A T A A A G A T G A T G T G A A T T A GT A G 690 — T T A T G T A A G A A T G T T T G A G A G A A A 691 — A G TA A A T G A T G A A T G A T A T G A T G A 692 — G A A A T T T G T G T TA A A G T T G A A T G A 693 — G A T G A A T G A T T G T G T T T A A G TA T A 694 — G A A A T A A G T G A G A G T T A A T G A A A T 695 — T G TT G A A A T A G T T A T T A G T T T G T G 696 — T T T G A G A G T A T AT T G A T A T G A G A A 697 — A T T G T G T G T A A A G T A A G A T T TA A G 698 — T A T A G T T T G A A G T G T G A T G T A T T T 699 — G T GA A G T T A T A G T G T A T A A A G A A T 700 — G T A T G T T G A A T AG T A A A T A G A T T G 701 — T T A G A A A G T G T G A T T T G T G T AT T T 702 — T T T A G T A A T A T G T A A G A G A T G T G A 703 — A G TA T G T A T A G A T G A T G T T T G T T T 704 — A T T T A A G T A A A GT G T A G A G A T A A G 705 20 A T T T G T G T T G A A T T G T A A A G TG A A 706 — A T G T T A T T A G A T T G T G A T G A A T G A 707 — T A GT A G T A G A A T A T G A A A T T A G A G 708 — T T T A A T G A G A A GA G T T A G A G T A T A 709 — A A A G T T T A G T A G A G T G T A T G TA A A 710 — A T A T A T G A T A G T A G A G T A G A T T A G 711 — T G AG A A G T T A A T T G T A T A G A T T G A 712 — T A T A G A G A T G T TA T A T G A A G T T G T 713 — A A A T T T G T T A A G T T G T T G T T GT T G 714 — T T G T T G A A G A T G A A A G T A G A A T T A 715 — A A GA G A T A A G T A G T G T T T A T G T T T 716 — A A T A A G A A G A A GT G A A A G A T T G A T 717 — T A A G T T A A A G T T G A T G A T T G AT A G 718 — A T A T A A G A T A A G A G T G T A A G T G A T 719 — G T TA A A T G T T G T T G T T T A A G T G A T 720 — G A G T T A A G T T A TT A G T T A A G A A G T 721 — T A T T A G A G T T T G A G A A T A A G TA G T 722 21 T A A T G T T G T T A T G T G T T A G A T G T T 723 — G A AA G T T G A T A G A A T G T A A T G T T T 724 — T G A T A G A T G A A TT G A T T G A T T A G T 725 — A T G A T A G A G T A A A G A A T A A G TT G T 726 — A G T A A G T G T T A G A T A G T A T T G A A T 727 27 A T GT A G A T T A A A G T A G T G T A T G T T 728 — T T A T T G A T A A T GA G A G A G T T A A A G 729 — A T T T G T T A T G A T A A A T G T G T AG T G 730 29 T T G A A G A A A T A A G A G T A A T A A G A G 731 — T G TG T A A T A A G T A G T A A G A T T A G A 732 — A T G A A A G T T A G AG T T T A T G A T A A G 733 37 A T T A G T T A A G A G A G T T T G T A GA T T 734 — T G T A G T A T T G T A T G A T T A A A G T G T 735 — A G TT G A T A A A G A A G A A G A G T A T A T 736 — G T A A T G A G A T A AA G A G A G A T A A T T 737 — T G T G T T G A A G A T A A A G T T T A TG A T 738 — A A G A A G A G T A G T T A G A A T T G A T T A 739 — G A AT G A A G A T G A A G T T T G T T A A T A 740 — A A A T T G T T G A G AT A A G A T A G T G A T 741 — T G A T T G T T T A A T G A T G T G T G AT T A 742 — A T G A A G T A T T G T T G A G T G A T T T A A 743 — G T GT A A A T G T T T G A G A T G T A T A T T 744 46 A A T T G A T G A G T TT A A A G A G T T G A T 745 — T T T G T G T A A T A T G A T T G A G A GT T T 746 — G T A G T A G A T G A T T A A G A A G A T A A A 747 — T T TA A T G T G A A A T T T G T T G T G A G T 748 — G T A A A G A A T T A GA T A A A G A G T G A T 749 — A A T A G T T A A G T T T A A G A G T T GT G T 750 — G T G T G A T G T T T A T A G A T T T G T T A T 751 — G T AT A G T G T G A T T A G A T T T G T A A A 752 49 G T T G T A A G A A A GA T A T G T A A G A A A 753 — A T A T T A G A T T G T A A A G A G A G TG A A 754 — G A G T G A T A T T G A A A T T A G A T T G T A 755 — T A AG A A G T T A A A G A A G A G A G T T T A 756 — G A T G T T A G A T A AA G T T T A A G T A G T 757 — G T G A T T G T A T G A G A A A T G T T AA A T 758 — T G A T T A T T G T A A G A A A G A T T G A G A 759 — A A GA A T T G T G T A A G T T T A T G A G T A 760 — T T G T A T T T A G A AG A T T T G T A G A T G 761 — T A T A T G T T T G T G T A A G A A G A AA T G 762 — G A T A A T G T G T G A A T T T G T G A A T A A 763 — T T AG A A A T G T G A G A T T T A A G A G T T 764 — A G T G T A G A A T T TG T A T T T A G T T G T 765 — T A G T T A A G A T A G A G T A A A T G AT A G 766 — G A A G T G A T A T T G T A A A T T G A T A A G 767 — G T AA T T G T G T T A G A T T T A A G A A G T 768 — T G A T A T T T G T G AA T T G A T A G T A T G 769 — A A G T A A A G A G A T A T A G T T A A GT T G 770 — A T T A G T T A A G T T A T T T G T G A G T G A 771 — A G AT G A A G T A G T T T A T G A A T T A G A 772 — T G A G T T A G T T A AG T G A T A G T T A A A 773 — T T A T T G T A G A T T T A G A G A A G AT G A 774 — T A T T T G T G T T T G T T G A T T A G A T A G 775 — G T AT A A T G T G T G T G A A A G T T A T A A 776 — T A T A T G T T G A G TA T A A A G A G A G A A 777 — T T A G T T A G T T T A A A G A T T G T GA G T 778 — T T T A G A A T A A G T G A T G T G A T G A A A 779 — A G AG T A A T G T G T A A A T A G T T A G A T 780 — T G T G A T A A A G A GA A A T T A G T T G T T 781 — G A A T T T A G T G A A T G T T T G A G AT T A 782 — T G T G A T G T G T A A G T A T A T G A A A T T 783 — T T GT G A A T G A T T A A T G A A T A G A A G 784 51 A A T G T T G T T T A GA T T G A G A A A G T T 785 — A G A T T G T G T T A G T A T T A G T A TA A G 786 — T T G A T G T A T T A G A A A G T T T A T G T G 787 — T A TG A T T G T G T G T T A G A G A A T T T A 788 — T A G T G T A G A T A TT T G A T A G T T A T G 789 52 A G T T T A A T G T G T T T A G T T G T TA T G 790 — T G T G T A A A G T A G A A A G T A A A G A T T 791 — G T TA T G A T A T A G T G A G T T G T T A T T 792 53 T T T G A T T G A A T GT T A A T A G T G T G T 793 — A G A G T A T T A G T A G T T A T T G T AA G T 794 54 T A A G T A G A A A G A A G A A G A T A T T T G 795 — A G AA A G A G A A T T A T G T A A T G A A A G 796 — T T A G A T T T G T T AG T G T G A T T T A A G 797 — G A T G A T T A A G A T A T A G A G A T AG T T 798 — A T A T T T G A G T G A T T A A G A G T A A T G 799 — T G TA T T G T G A G T T A A G T A T A A G T T 800 — A A T T T A G T A G A AA G T G T T G T G T T T 801 — G T T A G A A G A T T A A G T T G A A T AA T G 802 — T A A A G T A T G T G A G A T G A T T T A T G T 803 — T G AA A T G A T T A A A G A T G A A G A T G A 804 — T T A T T A G A T G T TG A G T G T T T G T T T 805 — T A G T G T T T A A A G A G T A G T A T AT G A 806 — A G T T A T A A G T A A A T G A T G T T G A T G 807 — T T AA G A G A G A A A T A A G T G T A T T G T 808 — G A T A T T G A A A T GT G T A A A T G A T G A 809 — A T G A T G A A T T A A G A A A G A A A GA G A 810 — G A A T A G T T T G A T T T G T G T T T G T T A 811 — A G TT G T T T A G A T T T G A T T T G T A A G 812 — G T A T G A G A T T T GA T A T A A G A T T A G 813 — T T T A T A G T G A G T A T A G T G A T GA T T 814 — T A T A T G T G A A G A T A T A A G T G T T T G 815 — A T TG A T A G A T G A T A G T A A T T G A G T 816 — T G A T A G A T G T G AA G A A T T T G A T T T 817 — G A A G A T A T T G A A A G A A T T T G AT G T 818 55 G A T G T T T A G T G T A G A T A T A G A T T T 819 — G A AT A T T G A G T T A T A A G T A G T A G T 820 — A G T G A G T A A G T AA T A G A A A G A T T T 821 — G T A G A A T A A G T A A T T T G T G A GA T A 822 — G A G T T A T T T G A G A T T T A G A T G T T T 823 — G A AA T G A T G A T T G A A T T T A G A G A T 824 — A A A T A G T G T G A GA A T A G T T A A G T A 825 — A T G T G T T A A G T T G T A G A A G A AT A A 826 — A T A A T G A G T T A A T A G T G T A A G A A G 827 — A T AA G A G A T G T T T A A G T T A G A A A G 828 — T G T T A G T G T T A GA A A T A T G A A A G A 829 — T T T A G A A G A T T G T T A G A T A A GT T G 830 — G T G T A A T G T A T A A G A T A G T T A A G T 831 — T A TT A G A G A G A A A T T G T A G A G A T T 832 57 T A G T G A G A T A A AG T A A A G T T T A T G 833 — T T G T G A A A G T T A A G T A A G T T AG T T 834 — A A A G T G T A A G T T G A A G A A T A T T G A 835 — G A AT A G A G T G T T A T T T G A A A T A G A 836 — T A T A A G A G A G A GA T A A G T A A T A A G 837 — T G A G T G A A A T T G A T A G A G T A AA T T 838 — G A T G A A T A A G T T T A A G T G A G A A A T 839 — G T GT G A T A T G T T T A T T G A T T A A G T 840 — T A A A G T G A G T G TA A A T G A T A A T G A 841 — G T A G A G T T T G A T T T G A A A G A AT A T 842 — G A A T A T T G T T A T G T T T G T T A T G A G 843 — G T GT A A T A A G A T G T A T T G T T G T T T 844 — T A A A T T G A T T G TG A G T T G A A G A A T 845 — T G A G A T A G T T A T A G T T A A G T TT A G 846 — A G T T T G T T A A G A T T A T G T A G A A A G 847 — G A AT G T G T A G A A T A A G A G A T T A A A 848 — G T A T T A T G A A A GA A G T T G T T G T T T 849 — G T G T T A T A G A A G T T A A A T G T TA A G 850 58 T T A A G A G T A G T G A A T A T G A T A G T A 851 — A A TG T T A T A A G A T G A G A G T T T A G T 852 — A T A T A A G A T T T GA T G T A G T G T A G T 853 — T A T G T T T G T T G T T G T T A A G T TT G A 854 — G A T A G T T T A G T A T A G A A G A T A A A G 855 — G T TG A A T A T A G A G A T A G T A A A T A G 856 — A G A G A A G A T T T AG T A A G A A T G A T A 857 — T G A A T G A G A A A G A T A T T G A G TA T T 858 — T G A A G A T T A T A G T A G T T G T A T A G A 859 — G A TT A G T A G T A T T G A A G A T T A T G T 860 — T G A A A T G T G T A TT T G T A T G T T T A G 861 59 A T T A A A G T T G A T A T G A A A G A AG T G 862 — A A T G T A G A G A T T G T A G T G A A T A T T 863 62 T T AT T T G T T G A G T G T A A A T G T G A T 864 — A T G T A A T T G T G AA T A A T G T A T G T G 865 63 G A T T T G T A T A G A G A T T A G T A AG T A 866 — A A T A T T G T T G T T T A G A G A A A G A A G 867 — A T GA T G A T G T A T T T G T A A A G A G T A 868 — A A T G T A T T T G T GT G A T T G T G T A A A 869 — A G T G T T A T G A A G A A T A G T A A GA A T 870 — G T T A T G T A G A G A T G A A A G A A A T T A 871 65 G T TT G T A T T A G A T A A A T G A G T T G T 872 — T G A T T T A T G A G AT T A A G A G A A A G A 873 — T T T G T G T G T T A T T G T A A T T G AG A T 874 70 G A T G T G T G A T A T G A T T A A A G A A A T 875 — A G AT T A T A G A T T T G T A G A G A A A G T 876 — G A A G A G T A T G T AA T A G T A T T G T A T 877 — T T T G T A A T G T T G T T G A G T T T AA G A 878 — A G T A A A T A G T A G T A T G A A T A A G A G 879 — G A AT G T T G A A T T G A A A T A T G A G T T 880 — A G T A G T T A A T T GA T A G T A A G T T T G 881 — A G T G T A A A G A A A T G A A T G A A TA A G 882 — T G T T A G A T A T T T G T G A A A T G T G A A 883 — T G TA T G T T G A G T T T G A A T T G T T A T 884 — T G A G T G A A T T A GT T A T G T T G T T A T 885 — G A A G A A A G A A A T G A G A A A G A TT A T 886 — T T A A G T A A G T T G T G T T G A T A T T A G 887 — A T GA T G T G T T T G A T T T G A A T T G A A 888 72 A A G T A A G T G A A AT T G T T G T T T G A A 889 — A T G A A G T G T A A A G T T T G A A A GA A A 890 — A G A G A G T A A G A T A A T T G T A T A G T A 891 — T T TA T G A G A T A G A T G A A A T A A G T G 892 — A G A A A T T A G T A GT A A T G A T T T G T G 893 — G A T T T G A G A T T G A A T G A G A A TA T A 894 — G A T T A G A A A G A T G A A T A A A G A T G A 895 — T A GA T A G A A A G T A T A T G T T G T A G T 896 — G A A G A T A G T A A AG T A A A G T A A G T T 897 — A A A T G T G T G T T T A G T A G T T G TA A A 898 75 T T G T T G A A G T A A G A G A T G A A T A A A 899 — T A TT T G A G A G A A A G A A A G A G T T T A 900 — T A T T T A G T G A T GA A T T T G T G A T G T 901 — T T A T A G T G A T G A T G A T A A G T TG A T 902 — T A A A G A T A A T T G T A G A A A G T A G T G 903 — G T TT A G T A T T G A T A T T G T G T G T A A 904 — G T G T T G T G A A T AA G A T T G A A A T A T 905 — A A A G A A A G T A T A A A G T G A G A TA G A 906 — T A T T T G T A A G A A G T G T A G A T A T T G 907 — T A GA A G A T G A A A T T G T G A T T T G T T 908 — A T A A T A G T A A G TG A A T G A T G A G A T 909 — A A T G T G A A T A A G A T A A A G T G TG T A 910 — A T T G A A G A T A A A G A T G T T G T T T A G 911 — T G AA A T A G A A G T G A G A T T A T A G T A 912 76 A G T T A T T G T G A AA G A G T T T A T G A T 913 — A A A T A G T A G T G A T A G A G A A G AT T T 914 — A G T G T A T G A A G T G T A A T A A G A T T A 915 — T G AT T A A G A T T G T G T A G T G T T A T A 916 — A G T T T A T G A T A TT T G T A G A T G A G T 917 — T A T G T G T A T G A A G A T T A T A G TT A G 918 78 G A A A T T G T T G T A T A G A G T G A T A T A 919 — T A GA A A T A G T T T A A G T A T A G T G T G 920 — T G A T T T A G A T G TT T A T T G T G A G A A 921 — A A G T T G A T A T T T G T T G T T A G AT G A 922 — T G A T G T G A T A A T G A G A A T A A A G A A 923 79 A A AG T T T A G T T T G T A T T A G T A G A G 924 — A G T T T G A T G T G AT A G T A A A T A G A A 925 — A A G T G T T A T T G A A T G T G A T G TT A T 926 — A A A T T G A A G T G T G A T A A T G T T T G T 927 — G T TT A G T G A T T A A A G A T A G A T T A G 928 82 A T A A G T G T A T A AG A G A A G T G T T A A 929 — A T G A A T T T G T T T G T G A T G A A GT T A 930 — A A A G A A T T G A G A A A T G A A A G T T A G 931 — A G TG T A A G A G T A T A A A G T A T T T G A 932 — G A A T T A A G A T T GT T A T A T G T G A G T 933 — T A T G A A A G T G T T G T T T A A G T AA G A 934 — T A A A G T A A A T G T T A T G T G A G A G A A 935 — A A AG A T A T T G A T T G A G A T A G A G T T 936 — A A G T G A T A T G A AT A T G T G A G A A A T 937 — A A A T A G A G T T T G T T A A T G T A AG T G 938 — G A T T T A G A T G A G T T A A G A A T T T A G 939 — T T GT A A A T G A G T G T G A A T A T T G T A 940 — A G T A G T G T A T T TG A G A T A A T A G A A 941 — T G A G T T A A A G A G T T G T T G A T AT T T 942 — A A A G A G T G T A T T A G A A A T A G T T T G 943 — G T TT A G T T A T T T G A T G A G A T A A T G 944 — A A G T G T A A A T G AA T A A A G A G T T G T 945 — A A T A A A G T G A G T A G A A G T G T AA T T 946 — T A T T G A G T T T G T G T A A A G A A G A T A 947 — T T TA T A G T T G T T G T G T T G A A A G T T 948 — A T G A A A T A T G A TT G T G T T T G T T G T 949 — A A A G A G A T G T A A A G T G A G T T AT T A 950 — T T G A A G A A A G T T A G A T G A T G A A T T 951 — A T GT T A T T T G T T T A G T T T G T G T G A 952 — A A A T A T G A A T T TG A A G A G A A G T G A 953 — G A T T A G A T A T A G A A T A T T G A AG A G 954 — T T A G A A T A A G A G A A A T G T A T G T G T 955 — T T TA T G A A A G A G A A G T G T A T T A T G 956 — G T A A G T A T T A A GT G T G A T T T A G T A 957 — A T A A A G A G A A G T A A A G A G T A AA G T 958 — A T T G T T A A T T G A A G T G T A T G A A A G 959 — T A TA T A G T T G A G T T G A G T A A G A T T 960 — T A G A T G A G A T A TA T G A A A G A T A G T 961 — A T A A G A A G A T G A T T T G T G T A AA T G 962 — T T A G T A A T A A G A A A G A T G A A G A G A 963 — G A TT T G T G A G T A A A G T A A A T A G A A 964 — A A A T A G A T G T A GA A T T T G T G T G T T 965 — G A A A T T A G T G T T T G T G T G T A TT A T 966 — A T T T G A G T A T G A T A G A A G A T T G T T 967 — A T AG A G T T G A A G T A T G T A A A G T T T 968 — T A A T T T G T G A A TG T T G T T A T T G T G 969 — T T A G T T T A T G A G A G T G A G A T TT A A 970 — G T T G T T A G A G T G T T T A T G A A A T T T 971 — T T TA T T G T G A T G T G A A A T A A G A G A 972 — G T A A G T A A T A T GA T A G T G A T T A A G 973 — T G A G A T G A T G T A T A T G T A G T AA T A 974 — A A T T G A G A A A G A G A T A A A T G A T A G 975 85 T T TG A A G T G A T G T T A G A A T G T T T A 976 — A G T T G T T G T G T AA T T G T T A G T A A A 977 — A T A G T G A G A A G T G A T A A G A T AT T T 978 — G T G T G A T A A G T A A T T G A G T T A A A T 979 — T A GT T A T T G T T T G T G A A T T T G A G A 980 — A T A G T T G A A T A GT A A T T T G A A G A G 981 — A T G T T T G T G T T T G A A T A G A G AA T A 982 — T G A T A A A G A T A T G A G A G A T T G T A A 983 — T A AA G A T G A G A T G T T G T T A A A G T T 984 — A A G T G A A A T T T GT A A G A A T T A G T G 985 — G A A A T G A G A G T T A T T G A T A G TT T A 986 — T T T G T A A A T G A G A T A T A G T G T T A G 987 — G T TA A T T G T G A T A T T T G A T T A G T G 988 — A G A G T G T T G A T AA A G A T G T T T A T A 989 — A A T T G T G A G A A A T T G A T A A G AA G A 990 — T T A A A G A G A A T T G A G A A G A G A A A T 991 — T T GT T A G A A G A A T T G A A T G T A T G T 992 — A G T T A A G A T A T GT G T G A T G T T T A A 993 — T G A G T T A T G T T G T A A T A G A A AT T G 994 — T T A G A T A A G T T T A G A G A T T G A G A A 995 — A T GA G T A A T A A G A G T A T T T G A A G T 996 — T G T T T A A G T G T AA T G A T T T G T T A G 997 — T T G A A G A A G A T T G T T A T T G T TG A A 998 — T A T A G A A A G A T T A A A G A G T G A A T G 999 — T A AA T T G T T A G A A A T T T G A G T G T G 1000 — A T T G T T A G T G T GT T A T T G A T T A T G 1001 — G A G A A T T A T G T G T G A A T A T A GA A A 1002 — T T G A T T G A T A A A G T A A A G A G T G T A 1003 — G TG T G T A A A T T G A A T A T G T T A A T G 1004 — A A A G T A A A G A AA G A A G T T T G A A A G 1005 — T T T A G T T G A A G A A T A G A A A GA A A G 1006 — G T G T A A T A A G A G T G A A T A G T A A T T 1007 — TA T T G A A A T A A G A G A G A T T T G T G A 1008 — A T G A G A A A G AA G A A G T T A A G A T T T 1009 — A A G A G T G A G T A T A T T G T T AA A G A A 1010 — T T T G T A A A G T G A T G A T G T A A G A T A 1011 —G A T G T T A T G T G A T G A A A T A T G T A T 1012 — G T A G A A T A AA G T G T T A A A G T G T T A 1013 — A A A G A G T A T G T G T G T A T GA T A T T T 1014 — A A A G A T A A G A G T T A G T A A A T T G T G 1015— A A G A A T T A G A G A A T A A G T G T G A T A 1016 — G A T A A G A AA G T G A A A T G T A A A T T G 1017 86 G A T G A A A G A T G T T T A AA G T T T G T T 1018 — A G T G T A A G T A A T A A G T T T G A G A A A1019 — G T T G A G A A T T A G A A T T T G A T A A A G 1020 87 T T A A GA A A T T T G T A T G T G T T G T T G 1021 — A G A A G A T T T A G A T GA A A T G A G T T T 1022 — T A A G T T T G A G A T A A A G A T G A T A TG 1023 — T G A G A T A G T T T G T A A T A T G T T T G T 1024 — A G T TT G A A A T T G T A A G T T T G A T G A 1025 — T A G A A T T G A T T A AT G A T G A G T A G T 1026 — A G A G A T T T G T A A T A A G T A T T G AA G 1027 — A T A A T G A T G T A A T G T A A G T A G T G T 1028 — T G AA A T T T G A T G A G A G A T A T G T T A 1029 — T G T G T A A A G T A TA G T T T A T G T T A G 1030 — T G A A T A A G T G A A A T A G A A T G AA T G 1031 — A A A G A A A G A T T G T A A T A A G T A G A G 1032 — A AT G A A A T A G T G T T A A A T G A G T G T 1033 89 G T A G A T A A A GA T G T G A A T T A T G A T 1034 — G A T A G T A T A T G T G T G T A T TT G T T T 1035 — A T G T T T G T A G A A A T G T T T G A A G A T 1036 —A A A T T T G T A G A G A G A A A T T T G T T G 1037 — T A G A A T A A GA T T A G T A A G T G T A G A 1038 — T G A T T T A G A G A A A T A T G AG T A G A A 1039 — A A T A G A G T A T G T T G T T T A T G A G A A 1040— G A T G A T G A A G A G T T T A T T G T A A A T 1041 — A A G T A A A GA A G A A G A A A T G T G T T A 1042 — T T G A A G A A T T A A G T G T TT A G T G T A 1043 — A G A A A G A A T G T T G A T T T A T G A T G T1044 — G A T T A A A G A G A T G T T G A T T G A A A T 1045 — A A T G AT A A T T G T T G A G A G A G T A A T 1046 — G T T T G T T G A A A G T GT A A A G T A T A T 1047 90 T G A G T T A T A T G A G A A A G T G T A AT T 1048 — T T G T G A G A A A G A A G T A T A T A G A A T 1049 — G T AA G T T T A G A G T T A T A G A G T T T A 1050 — G A T A G A T A G A T AA G T T A A T T G A A G 1051 — A G A G A T G A T T G T T T A T G T A T TA T G 1052 — A A A G T T A A G A A A T T G T A G T G A T A G 1053 — T TT G A T A T T G T T T G T G A G T G T A T A 1054 — A T T T G T A G A A AG T T G T T A T G A G T T 1055 — G A T T T G A G T A A G T T T A T A G AT G A A 1056 — A A G A T A A A G T G A G T T G A T T T A G A T 1057 — GA T A T T G T A A G A T A T G T T G T A A A G 1058 — G T A A G A G T G TA T T G T A A G T T A A T T 1059 — G T G T G A T T A G T A A T G A A G TA T T T A 1060 91 G T A A G A A A G A T T A A G T G T T A G T A A 1061 —A G T A G A A A G T T G A A A T T G A T T A T G 1062 92 T A A G A G A AG T T G A G T A A T G T A T T T 1063 — G T T A A G A A A T A G T A G A TA A G T G A A 1064 — T A A G T A A A T T G A A A G T G T A T A G T G1065 — A A G A T G T A T G T T T A T T G T T G T G T A 1066 — A T T T AG A A T A T A G T G A A G A G A T A G 1067 — G T T A T G A A A G A G T AT G T G T T A A A T 1068 93 T A T T A T G T G A A G A A G A A T G A T TA G 1069 — T A A T A A G T T G A A G A G A A T T G T T G T 1070 — T G AT G T T T G A T G T A A T T G T T A A A G 1071 — G T G A A A G A T T T GA G T T T G T A T A A T 1072 — A G A G A A T A T A G A T T G A G A T T TG T T 1073 — T T T G A G A T G T G A T G A T A A A G T T A A 1074 — G TT G T A A A T T G T A G T A A A G A A G T A 1075 94 G T G T T A T G A TG T T G T T T G T A T T A T 1076 — A T T A T T G T G T A G A T G T A T TA A G A G 1077 — G T T A G A A A G A T T T A G A A G T T A G T T 1078 —T T G T G T A T T A A G A G A G T G A A A T A T 1079 — G T T T A A G A TA G A A A G A G T G A T T T A 1080 — A A T G A G A A A T A G A T A G T TA T T G T G 1081 — T G A A T T G A A T A A G A A T T T G T T G T G 108295 A A T A A G A T T G A A T T A G T G A G T A A G 1083 — A A T G T T TG A G A G A T T T A G T A A A G A 1084 — A G T T T A G A A T A G A A A TG T G T T T G A 1085 — T A T A A G T A A G T G T T A A G A T T T G A G1086 — G T A G T G A A T A A G T T A G T G T T A A T A 1087 — A A G T GT G T T A A A G T A A A T G T A G A T 1088 — A G A G A T G T T T A T G TT G T G A A T T A A 1089 — A G T T G A A T A T T G A T G A T A A G A A GA 1090 — T G A A T G T G A G A T G T T T A G A A T A A T 1091 — A A T AA T G A T G T A A G T T T G A G T T T G 1092 — A A A G A G T G A A T A GA A A T A A G A G A A 1093 — A A T A A A G T T A T T G A G A G A G T T TA G 1094 — A G T A G T G T T G T A G T T T A G T A T A T A 1095 — G T AA G A A T G T A T T A G A T A T T T G T G 1096 — G A T A A A T G T T T GA T A A A G T A G T T G 1097 — A T A G T A T G T A T G T G T G A A G A TT T A 1098 — A T G A A T G T A G A G T G A T T A G T T T A A 1099 — G TA G T A T T T A G T G A T G T A A G A A T A 1100 — A G A A T T G T A T TG A A G A A G A A T A T G 1101 — T T T A T A G A A T T G A G A G A A G TT A A G 1102 — A A A G T A G T A G A G A T T T G A G A A T T A 1103 — TT T A A A G A A A G T A T T G T A A G A G T G 1104 — A A A T T G A G A AA G T G A A T G A A G T T T 1105 — A A G A A A T A A G T A T G A T A G TA G T A G 1106 — A T T T G A A T T G T A T T G T A G T T T G T G 1107 —A A G A G A A T A A T G T A G A G A T A T A A G 1108 — T G T G T A A T AG T T G T T A A T G A G T A A 1109 — T A T A G T T G T A G T T T A G A TG A A T G T 1110 — A T T G T G T T A G A A T G A T G T T A A T A G 1111— G T T T G T A T A G T A T T T G A T T G A T G T 1112 — A G A G T A A AG T A T G A G T T A T G A A T A 1113 — G A A A G T T T A A G T G A T G TA T A T T G T 1114 96 T T A A A T G A T A A A G A G T A G T G A A G T1115 — T T A A A T G T G T G A G A A G A T G A A T A A 1116 — A T T T GT A T A A A G T G A A G A A G A G A A 1117 97 T G A T T A G T A T T T GT G A A G A G A T T T 1118 — T T T G A A T G A A A T T G A T G A T A G AT G 1119 — A G A G T A A G A T T A A G A A T A A G A A A G 1120 — A T TG A A T T G A G A A G T G A A G T A A A T 1121 — T T T A G A G A A G T AT T G T T T G A A A G A 1122 — T A A A G T G A A A G A T T T G A A A T GA T G 1123 — G A A A G T T A G A G A A A T G T A G A A A T T 1124 — G TG A A T A A T G A A G A A G T T A T G T T A 1125 98 T T G T G A A T A AA G T A G A T G T G T T A T 1126 — T T A T A T G A T A T G A G T T T G TG T T G A 1127 — T T G A T T T G T G T G A G T A T T A G T T A T 1128 —A A A G T G A T T A A G T T A G T T T G A G A T 1129 — T T G T A T T T GT A T A A T G T T G A A G A G 1130 — G T T T G A A A T T A G T G T G A GA A A T A T 1131 — A A T G T T G A G A T T G A T A A T G T T G A A 1132— T A G T A G T A G T A T T G T T G T A A T A A G 1133 — G T T G T A A TT T G A G T G T T A G T T A T T 1134 — T G A A T A T G A T A G T T A G TA A T T G T G 1135 — T G A T A G T A T G T T T G T G A T T A A A G A1136 — G A T G T A T A A A G A G T A T G T T A T A A G 1137 — A G T G AG A T T T A G A A G A T G T T A T T A 1138 — A T G A G A A T T T G T T AA A G A G A A A G T 1139 — A A A G A A T T A G T A T G A T A G A T G A GA 1140 99 T A G A G T T G T A T A G T T T A T A G T T G A 1141 — G T A GA A T G A T T G T T T A G A A G A T T T 1142 — G T T T A T G T T T G A GA A G A G T T A T T T 1143 — T A G A A G T T T G A A A G T T A T T G A TT G 1144 — G A T G A A G A G T A T T T G T T A T A T G T A 1145 — G A TG A A T A T A G T A A G T A T T G A G T A 1146 100  T A G T G A T G A AA T T T G A G A T A G A T A 1147 — G A A A G A A A T T G A A G A G T T TG A T A T 1148 — A T T T G A G T A T T T G T G T A T T G A A T G 1149 —A T G A G T T G A A A T T T G A A G T A T T G T 1150 — T T A A T A G T GA G A G A G T A T A T G T A A 1151 — A T T A A G A G A G T G A G T A A AT G T A A A 1152 — A A G A A T A G A T G A G A T T A G A A A T A G 1153— A G T T T A A A G A G T T A G A A T T G A A A G 1154 — G T A A G A T TT G T T G A A T A A A G A A G A 1155 — A G A G A A A G A A G T T A A A GT G A T A T T 1156 — T A A T A G A G A A G A G A T G T A T G A A T A1157 — T T A T T A G T G A T A A G T G A A G T T T A G 1158 — A T A A TG T A A A G A T G A G T T T A T G A G 1159 — T T G A T T T G A G A G T TG A T A A G A T T T 1160 — A T G A T T A T T G T G T G T A G A A T T A GA 1161 — T A T A A A G A T A T A G T A G A T G A T G T G 1162 — T T T AG T T G A G A T G A A G T T A T T A G A 1163 — A T T G A A T T G A T A TA G T G T A A A G T G 1164 — G A A G A A A G A T T A T T G T A T T G A GT T 1165 — A T T G A G T G T A G T G A T T T A G A A A T A 1166 — A A TA A A G T G T T T A A G A G T A G A G T A 1167 — G T A G A G A T A A T TG A T G T G T A A T T T 1168 —

All references referred to in this specification are incorporated hereinby reference.

The scope of protection sought for the invention described herein isdefined by the appended claims. It will also be understood that anyelements recited above or in the claims, can be combined with theelements of any claim. In particular, elements of a dependent claim canbe combined with any element of a claim from which it depends, or withany other compatible element of the invention.

The invention claimed is:
 1. A composition comprising oligonucleotides,each oligonucleotide in the composition either being free of cytosineresidues and comprising guanosine residues or being free of guanosineresidues and comprising cytosine residues, wherein for eacholigonucleotide that comprises cytosine residues, the cytosine residuesare separated by between one and six non-cytosine residues, and for eacholigonucleotide that comprises guanosine residues, the guanosineresidues are separated by between one and six non-guanosine residues,wherein the number of cytosine and guanosine residues present in eacholigonucleotide in the composition does not exceed L/4 where L is thenumber of bases in the oligonucleotide, wherein the length of eacholigonucleotide in the composition differs by no more than five basesfrom the average length of all oligonucleotides in the composition,wherein each oligonucleotide in the composition contains no more than 4contiguous identical nucleotides, wherein the number of guanosine andcytosine residues present in each oligonucleotide in the compositiondoes not vary from the average number of guanosine and cytosine residuespresent in all other oligonucleotides of the composition by more thanone, and when each oligonucleotide of the composition is exposed tohybridization conditions comprising 0.2M NaCl, 0.1M Tris, 0.08% TritonX-100, pH 8.0 at 37° C., the degree of cross-hybridization between theoligonucleotide and any complement of a different oligonucleotide of thecomposition does not exceed 30% of the degree of hybridization betweenthe oligonucleotide and an oligonucleotide fully complementary thereto.2. The composition of claim 1, further characterized in that, eacholigonucleotide in the composition contains either a cytosine orguanosine residue located within seven residues of an end of theoligonucleotide.
 3. The composition of claim 1, wherein the length ofeach oligonucleotide in the composition is identical.
 4. The compositionof claim 3, wherein the number of guanosine and cytosine residuespresent in each oligonucleotide is the same.
 5. The composition of claim1, wherein the oligonucleotides are attached to a solid phase support.6. The composition of claim 5, wherein the support is a planar substratecomprising a plurality of spatially addressable regions.
 7. Thecomposition of claim 1, wherein the oligonucleotides are covalentlylinked to microparticles.
 8. The composition of claim 7, wherein themicroparticles are spectrophotometrically unique and each uniquemicroparticle has a different oligonucleotide attached thereto.
 9. Thecomposition of claim 1, wherein the degree of cross-hybridizationbetween a said oligonucleotide and any complement of a differentoligonucleotide of the composition does not exceed 10% of the degree ofhybridization between said oligonucleotide and a complement to saidoligonucleotide.
 10. The composition of claim 1, wherein eacholigonucleotide of the composition is at least ten nucleotides inlength.
 11. A composition comprising oligonucleotides, eacholigonucleotide of the composition consisting of a sequence of ten tofifty nucleotide bases in length and for which, under a single set ofhybridization conditions, the degree of cross-hybridization between asaid oligonucleotide and any complement of a different oligonucleotideof the composition does not exceed about 20% of the degree ofhybridization between said oligonucleotide and a complement to saidoligonucleotide, and wherein each sequence is free of cytosine residuesand comprises guanosine residues and, for each sequence, the number ofguanosine residues does not exceed L/4 where L is the number of bases insaid sequence.
 12. The composition of claim 11, wherein each saidsequence is of the same length as every other said sequence.
 13. Thecomposition of claim 12, wherein each said sequence is twenty-four basesin length.
 14. The composition of claim 11, wherein the number ofguanosine residues in each said sequence does not vary from the averagenumber of guanosine residues in all of the sequences of the set by morethan one.
 15. The composition of claim 14, wherein each said sequence istwenty-four bases in length and each said sequence contains sixguanosine residues.
 16. The composition of claim 11, wherein at the5′-end of each said sequence at least one of the first, second, third,fourth, fifth, sixth and seventh bases of the sequence is a guanosineresidue.
 17. The composition of claim 11, wherein at the 3′-end of eachsaid sequence at least one of the first, second, third, fourth, fifth,sixth and seventh bases of the sequence is a guanosine residue.
 18. Thecomposition of claim 16, wherein at the 3′-end of each said sequence atleast one of the first, second, third, fourth, fifth, sixth and seventhbases of the sequence is a guanosine residue.
 19. The composition ofclaim 11, wherein each said oligonucleotide is linked to a solid phasesupport so as to be distinguishable from a mixture of other saidoligonucleotides by hybridization to its complement.
 20. The compositionof claim 19, wherein each oligonucleotide is linked to a location on asaid solid phase support that is different than the location for eachother different said oligonucleotide.
 21. The composition of claim 20,wherein each said solid phase support is a microparticle and each saidoligonucleotide is covalently linked to a different microparticle thaneach other different said molecule oligonucleotide.
 22. The compositioncomprising oligonucleotides, each oligonucleotide of the compositionconsisting of a sequence of ten to fifty nucleotide bases in length andfor which, under a single set of hybridization conditions, the degree ofcross-hybridization between a said oligonucleotide and any complement ofa different oligonucleotide does not exceed about 20% of the degree ofhybridization between said oligonucleotide and a complement to saidoligonucleotide, and wherein each sequence is free of guanosine residuesand comprises cytosine residues and, for each sequence, the number ofcytosine residues does not exceed L/4 where L is the number of bases insaid sequence.
 23. The composition of claim 22, wherein each saidsequence is of the same length as every other said sequence.
 24. Thecomposition of claim 23, wherein each said sequence is twenty-four basesin length.
 25. The composition of claim 22, wherein the number ofcytosine residues in each said sequence does not vary from the averagenumber of cytosine residues in all of the sequences of the set by morethan one.
 26. The composition of claim 25, wherein each said sequence istwenty-four bases in length and each said sequence contains six cytosineresidues.
 27. The composition of claim 22, wherein at the 5′-end of eachsaid sequence at least one of the first, second, third, fourth, fifth,sixth and seventh bases of the sequence is a cytosine residue.
 28. Thecomposition of claim 22, wherein at the 3′-end of each said sequence atleast one of the first, second, third, fourth, fifth, sixth and seventhbases of the sequence is a cytosine residue.
 29. The composition ofclaim 27, wherein at the 3′-end of each said sequence at least one ofthe first, second, third, fourth, fifth, sixth and seventh bases of thesequence is a cytosine residue.
 30. A composition of claim 22, whereineach said oligonucleotide is linked to a solid phase support so as to bedistinguishable from a mixture of other said oligonucleotides byhybridization to its complement.
 31. The composition of claim 30,wherein each oligonucleotide is linked to a location on a said solidphase support that is different than the location for each otherdifferent said oligonucleotide.
 32. The composition of claim 31, whereineach said solid phase support is a microparticle and each saidoligonucleotide is covalently linked to a different microparticle thaneach other different said oligonucleotide.
 33. A composition comprisingoligonucleotides each attached to a solid support, each attachedoligonucleotide in the composition either being free of cytosineresidues and comprising guanosine residues or being free of guanosineresidues and comprising cytosine residues, wherein for each attachedoligonucleotide that comprises cytosine residues, the cytosine residuesare separated by between one and six non-cytosine residues, and for eachattached oligonucleotide that comprises guanosine residues, theguanosine residues are separated by between one and six non-guanosineresidues, wherein the number of cytosine and guanosine residues presentin each attached oligonucleotide in the composition does not exceed L/4where L is the number of bases in the oligonucleotide, wherein thelength of each attached oligonucleotide in the composition differs by nomore than five bases from the average length of all attachedoligonucleotides in the composition, wherein each attachedoligonucleotide in the composition contains no more than 4 contiguousidentical nucleotides, wherein the number of guanosine and cytosineresidues present in each attached oligonucleotide in the compositiondoes not vary from the average number of guanosine and cytosine residuespresent in all other attached nucleotides of the composition by morethan one, and when each attached oligonucleotide of the composition isexposed to hybridization conditions comprising 0.2M NaCl, 0.1M Tris,0.08% Triton X-100, pH 8.0 at 37° C., the degree of cross-hybridizationbetween the attached oligonucleotide and an oligonucleotide of thecomposition that is not fully complementary to the attachedoligonucleotide does not exceed 30% of the degree of hybridizationbetween the attached oligonucleotide and a fully complementaryoligonucleotide to said attached oligonucleotide.
 34. The composition ofclaim 33, further characterized in that, each attached oligonucleotidein the composition contains either a cytosine or guanosine residuelocated within seven residues of an end of the oligonucleotide.
 35. Thecomposition of claim 33, wherein the length of each attachedoligonucleotide in the composition is identical.
 36. The composition ofclaim 35, wherein the number of guanosine and cytosine residues presentin each attached oligonucleotide is the same.
 37. The composition ofclaim 33, wherein the support is a planar substrate comprising aplurality of spatially addressable regions.
 38. The composition of claim33, wherein the attached oligonucleotides are covalently linked tomicroparticles.
 39. The composition of claim 38, wherein themicroparticles are spectrophotometrically unique and each uniquemicroparticle has a different oligonucleotide attached thereto.
 40. Thecomposition of claim 33, wherein the degree of cross-hybridizationbetween a said attached oligonucleotide and any complement of adifferent oligonucleotide of the composition does not exceed 10% of thedegree of hybridization between said attached oligonucleotide and acomplement to said attached oligonucleotide.
 41. The composition ofclaim 33, wherein each attached oligonucleotide of the composition is atleast ten nucleotides in length.